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.     

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.     

THE RABBIT ZYGOTE : III. Formation of the Blastomere Nucleus

The formation of the blastomere nucleus was examined in the rabbit zygote with the electron microscope. In late anaphase the chromosomes are bare and vesicles of the smooth endoplasmic reticulum are numerous in the vicinity of the chromosomes. In early telophase individual chromosomes attain their own nuclear envelope and they are called karyomeres. The envelope of the karyomeres contains small gaps within it at several places where the chromatin is exposed to the cytoplasm. Nuclear pores are also observed. In the cytoplasm short annulate lamellae appear adjacent to the karyomeres, and clusters of punctate substance are also present. From early telophase onward the karyomeres extend pseudopod-like structures, called karyopods, which extend toward other karyomeres or karyopods, and consequently fuse together and serve as chromosomal bridges. Eventually all of the karyomeres fuse into a dense nucleus and decondensation of the chromosomes occurs.     

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.     

Mitosis and microtubule organizational changes in isolated generative cells of Allemanda neriifolia

Summary The organizational changes of the microtubules of isolated generative cells of Allemanda neriifolia during division were followed using anti--tubulin and immunofluorescence microscopy. Generative cells were isolated from the pollen tubes after osmotic shock treatment. Immediately after isolation most of the cells remain either in early or late prophase. The shape of the cell changes from spindle to spheroidal. In early prophase the nuclear membrane of the cell appears intact and the cytoplasm full of reticulate microtubules of different shapes and thicknesses. Later, the nuclear membrane breaks up. After the nuclear membrane has broken up, the chromosomes scatter into the cytoplasm and mix with the microtubules. When cells enter metaphase, spindle microtubules form. Afterwards, in anaphase, sister chromatids separate and the spindle disappears. A new array of longitudinally oriented cage microtubules appears. As the cells enter early telophase, the cage microtubules disappear and an array of interpolar microtubules begins to form. Later, in some telophase cells the interpolar microtubules become highly elongated, but in others they soon disappear and become replaced by a thick band(s) (or sheet(s)) of microtubules in the midplane between the two clusters of chromosomes and the cell shape reverts back to spheroidal. In culture no phragmoplast junctions appear in any of the late telophase cells although they are present under the in situ condition (i.e. in pollen tubes).     

The centrosome cycle in syncytial Drosophila embryos analyzed by energy filtering transmission electron microscopy.

We have investigated the centrosome cycle in Drosophila syncytial embryos at the ultrastructural level by using a transmission electron microscope equipped with an electron energy filtering device (Omega filter). This new technique allows the study of uncontrasted thick sections with a high resolution. We have been able to characterize two classes of filamentous structures in the centrosomal apparatus that were not detectable on ultrathin sections. These new filamentous structures are: 1) a very orderly lattice that connects the two centrioles during mitosis; and 2) a fibrogranular connection between the centrosome and the nuclear envelope. The intercentriolar linkage could be involved in the precise timing of separation of the centrioles during late anaphase. The centrosome-nuclear envelope connection probably prevents the loss of centrosomes in this syncytial environment, and ensures the proper migration of the centrosomes along the surface of the nucleus. This connection may also couple the nuclei to the cytoskeleton, thus allowing their migration and their anchorage to the cortex at the blastoderm stage. This thick section analysis has also allowed us to precisely reconstitute the centrosome cycle. From their separation at telophase and throughout most of interphase, centrosomes are composed of a single centriole. We conclude that in the early Drosophila embryo there is an unusual delay between the separation of the parent centrioles and their duplication. This leaves a surprisingly short time to assemble a daughter centriole.     

Genetic Control of Mitosis: Mutation ff3Causes Premature Beginning of Telophase Chromatin Reorganization in Drosophila melanogaster

The effect of cell cycle mutation ff3 on chromosome segregation was studied on fixed cells of neural ganglia of Drosophila melanogasterlarvae. The cell distributions by diameter of interphase nuclei and by distance between sister chromatid sets were compared at anaphase and telophase. In the control wild-type strain Lausenne, the cell distribution by distance between sister chromatids in anaphase was similar to their distribution by nuclear size. The mean distance between segregating chromatids at anaphase (l _av) coincided with the mean diameter of interphase nuclei (d _av) and was 8.3 m. Cells passed to telophase when chromatids were at least 10 m apart. The mutant ff3 strain differed from the control strain Lausenne in cell distribution by interphase nuclear diameter and distance between sister chromatids in anaphase; the mean nuclear diameter and mean distance between segregating chromatids similarly increased to 9.3 m. A specific feature of mitosis in mutant strain ff3 was a premature beginning of telophase chromatin reorganization. This caused the occurrence of cells with abnormally short (less then the interphase nuclear diameter) distance between sister chromatid sets in telophase but not in anaphase, as if these cells had passed from anaphase to telophase prematurely, during the chromatid movement toward poles in anaphase A.     

Superficial Warming of Epoxy Blocks for Cutting of 25-150 μm Sections to be Resectioned in the 40-90 nm Range

An improved method is described in which tissue areas can be initially identified in thick sections by light microscopy and isolated for subsequent ultrathin sections and observation by electron microscopy. This is achieved by embedding in hard Epon which can be sectioned at 25-150 μm on a sliding microtome after softening the blockface by applying a 60-70 C tacking iron to its surface immediately before each section is taken. The thick sections are then mounted on plastic slides to enable light microscopic selection of areas to be observed by electron microscopy. The selected areas are remounted on faced Epon blanks and resectioned at less than 50 nm. This technique makes it possible to obtain thick sections while maintaining an Epon hard enough for good serial ultrathin sections.     

Morphometric analysis of several intracellular events occurring during the vegetative life cycle of the unicellular algaPolytoma papillatum

Summary Quantitative electron microscopy of serial sections was used to study thePolytoma papillatum cell and some of its constituents (nucleus, chondriome, leucoplast) during its vegetative life cycle.The volumes of cells just entering into or passing through mitosis varied considerably and seemed to determine the number of subsequent division processes.Whereas a volumetric balance existed between the cell (100%) and the chondriome (8–9%) during the whole life cycle, there was a correlation between cell and nuclear volumes (8–10%) only during interphase growth and the onset of mitosis. At telophase the nucleus-to-cell-volume ratio was reduced to 2%, but gradually increased during cytokinesis (4.6% at early cytokinesis; 6.5% at late cytokinesis) until it reached the initial value again in newly formed daughter cells. The leucoplast-to-cell-volume ratio (10–26%) varied considerably without any recognizable dependence upon cell cycling.The mean short axis of mitochondrial profiles was proportional to the mean diameter (=thickness) of the mitochondria; the specific surface (outer membrane area per 100 m³ mitochondrial volume), and the surface-to-volume ratio changed rhythmically. Changes in mitochondrial surface-to-volume ratio (Sc/Vc) were apparently correlated with changes in mitochondrial diameter (Dc). This relationship can be approximately described by the function Sc/Vc=4/Dc.Deviations of the surface-to-volume ratios of the nuclei from the surface-to-volume ratios of idealized spheres of equal size, indicating profound changes in nuclear shape, were found mainly during mitosis.Results were compared with those obtained from other morphometric investigations and discussed with regard to their functional meaning.     

Immunofluorescence and electron microscopic studies of microtubule organization during the cell cycle ofDictyota dichotoma (Phaeophyta,Dictyotales)

Summary Interphase cells ofDictyota dichotoma (Hudson) Lamour. lack cortical microtubules (Mts) but display an impressive network of cytoplasmic microtubules (c-Mts). These are focussed on two opposed perinuclear centriolar sites where centrin or a centrin-homologue is localized. Some of the Mts surround the nucleus, but the majority traverse the cytoplasm as bundles variously directed towards the plasmalemma. In apical cells, and to a lesser extent in the square or slightly elongated meristematic cells, Mts are more or less evenly arranged. In elongated cells they form thick bundles longitudinally traversing the cytoplasm; a pattern maintained in differentiated cells. In early prophase the non-perinuclear Mts disappear but by late prophase a bi-astral arrangement of short Mts is observed. They enter polar nuclear depressions and attach to differentiated regions of the nuclear envelope where polar gaps open. By metaphase the spindle Mts converge on the centrioles at the polar gaps. At anaphase, interzonal Mts are evident and the asters start to reassemble. After telophase disruption of the interzonal Mts, the daughter nuclei approach each other, but move apart again before cytokinesis. The latter movement keeps pace with the development of two interdigitating Mt systems, ensheathing both daughter nuclei. The partition membrane bisects this Mt cage. Between telophase and cytokinesis the centrosomes separate, finally occupying opposed perinuclear sites. New Mts arise at the new centrosomes, some terminating on the consolidating partition membrane. Our data show thatD. dichotoma vegetative cells display a prominent cytoplasmic Mt cytoskeleton, which undergoes continual, but definite, change in organization during the cell cycle.     

Large-scale chromatin structural domains within mitotic and interphase chromosomes in vivo and in vitro

Higher-order chromatin structural domains approximately 130 nm in width are observed as prominent components of both Drosophila melanogaster and human mitotic chromosomes using buffer conditions which preserve chromosome morphology as determined by light microscopic comparison with chromosomes within living cells. Spatially discrete chromatin structural domains of similar size also exist as prominent components within interphase nuclei prepared under equivalent conditions. Examination of chromosomes during the anaphase-telophase transition suggests that chromosomes decondense largely through the progressive straightening or uncoiling of these large-scale chromatin domains. A quantitative analysis of the size distribution of these higher-order domains in telophase nuclei indicated a mean width of 126±36 nm. Three-dimensional views using stereopairs of chromosomes and interphase nuclei from 0.5 m thick sections suggest that these large-scale chromatin domains consist of 30 nm fibers packed by tight folding into larger, linear, fiber-like elements. Reduction in vitro of either polyamine or divalent cation concentrations within two different buffer systems results in a loss of these large-scale domains, with no higher-order chromatin organization evident above the 20–30 nm fiber. Under these conditions the DNA distribution within mitotic chromosomes and interphase nuclei appears significantly diffuse relative to the appearance by light microscopy within living cells, or, by electron microscopy, within cells fixed directly without permeabilization in buffer. These results suggest that these large-scale chromatin structural domains are fundamental elements of chromosome architecture in vivo.     

RNA Polymerase B levels during the cell cycle ofPhysarum polycephalum

Summary Tritiated -amanitin has been used as a specific and sensitive probe to estimate the number of RNA polymerase B molecules in isolated nuclei, chromatin and nucleoids, obtained from macroplasmodia ofPhysarum polycephalum. During mitosis (metaphase±10 min) there is at least 10-fold less RNA polymerase B than at all phases of the cell cycle, even if DNA replication has been blocked in vivo. It is concluded that many of the RNA polymerase B molecules leave the chromatin during decondensation of the chromosomes in telophase of the synchronous nuclear division ofPhysarum.     

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.     

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.     

Ultrastructure of holokinetic mitotic chromosomes and interphase nuclei of Tetranychus urticae Koch (Acari,Tetranychidae) in relation to loss and missegregation of induced fragments

The association of microtubules with mitotic holokinetic chromosomes of Tetranychus urticae Koch was investigated in serial ultrathin sections. Reconstructions from 14 series showed that 60–100 microtubules were associated with the entire poleward surfaces of the chromosomes. In the telophase of early developmental stages the chromosomes were decondenseed into separate micronuclei, containing at least one nucleolus. From these morphologic data, the fate of induced chromosome fragments, described in earlier papers, is surmised to depend on events in interphase as well as in mitosis.     

Relationship between nucleoli and sex chromosomes during meiosis of the male wood lemming Myopus schisticolor: a fine-structure study

Electron microscopy of ultrathin serial sections has been used to study the origin and fate of a mass of fibrillar material (FM) during spermatogenesis in the wood lemming Myopus schisticolor. In the course of early pachytene, one of the two nucleoli completely disappears. The remaining nucleolus loses its granular portion and acquires a "round body" encased by the fibrillar moiety, and the restructuring is accompanied by the appearance of FM in the close vicinity of this nucleolus. During diakinesis, the FM increases in volume and density and selectively infiltrates the chromatin of the XY pair. The intermingling of sex chromosomes and FM is at its maximum in metaphase I, giving the XY chromatin a patchy appearance. The FM separates along with the chromatin during the ensuing anaphase I and is shed from the chromosomes during early telophase I. By the time the nuclear envelope is reconstituted, the FM is completely separated from the chromatin. It disintegrates in the spermatids. The FM could not be stained using the Ag-NOR technique. In the wood lemming, X and Y chromosomes show an end-to-end association without a detectable synaptonemal complex. The FM may contribute to the attachment of the two sex chromosomes to each other. Thus, the FM is considered to be a substitute for a chiasma, which normally guarantees proper segregation in anaphase I.     

Protein staining of ribboned epon sections for light microscopy

Summary Procedures are described for obtaining and handling ribboned epon sections 0.3–2 thick for light microscopy, and for the cytological application of two intense acid dyes, Aniline Blue Black and Coomassie Brilliant Blue R 250. The technique allows precise localization of proteins and some other materials, and, because the sections are ribboned, facilitates three-dimensional visualization of the structures involved. The dyes may be used in combination with the periodic acid-Schiff reaction and with autoradiography.This work was supported in part by a Public Health Service fellowship 5-F2-GM-22, 031-02 to the author and in part by NSF grant GB 3460 to Dr. W. A. Jensen.     

Synaptinemal complexes in the achiasmatic spermatogenesis of Bolbe nigra Giglio-Tos (Mantoidea)

In chiasmatic meiosis of mosquitoes, ascomycetes and lilies the synaptinemal complex (SC) disassociates from the bivalent before metaphase I. Conversely, in the achiasmatic meiosis of Bolbe nigra, the SC remains associated with the bivalent during first metaphase. Light microscopy reveals mid-bodies between disjoining half-bivalents during early first anaphase in Bolbe. Optically controlled serial sections for electron microscopy show that the mid-bodies seen in light micrographs and synaptinemal complexes seen in electron micrographs are the same structure. Electron micrographs indicate that the SC breaks transversely at a point corresponding to the chromosomal kinetochore during anaphase I as the chromatin and the SC begin to separate. During telophase I, SC remnants are at the poles with the chromosomes or between poles. Presently, the evidence is inadequate to state whether the SC serves alternately or simultaneously as a biological contrivance for conjunction and crossing-over or singly as a device for one of these phenomena.Supported by a University of Melbourne Research Fellowship.     

3H-actinomycin D binding to ultrathin section of plastic-embedded Locusta migratoria testicular tubules. Improvement of the technique and further characterization of the reaction

Semi-thin and ultrathin sections of locust testes have been incubated in 3H-actinomycin D solution and submitted to radioautography. The improved technical conditions described allow the reproducible obtainment of cell radioautographs with a moderate nuclear labelling and a very low nonspecific background which are usable for semi-quantitative results. Extraction with enzymes (DNase, RNase, pronase) or concentrated salt solution have been carried out before 3H-Actinomycin D treatment in order to characterize the reaction. The semi-quantitative results obtained at the light microscope level suggest that, in relation to the structural and chemical changes which occur in chromatin during spermiogenesis, some proteins may be easily hydrolysed in early spermatids. In ultrathin sections of spermatocytes the X chromosome is heavily "stained" with 3H-Actinomycin D, while 3H-uridine is not incorporated into the sex chromatin. These results are discussed in the light of current ideas on the constitution of active chromatin.     

Ultrastructural demonstration of cartilage acid glycosaminoglycans

Synopsis A method for the demonstration of cartilage acid glycosaminoglycans by light and electron microscopy is described. Rabbit ear cartilage was fixed in cacodylate buffered 2.5% methanol-free formaldehyde with 0.001 M Ruthenium Red andp-chloromercuribenzoate (PCMB). Dehydration was carried out in ethylene glycol followed by embedding in the water-soluble glycol methacrylate (GMA). In some experiments unfixed cartilage was rapidly dehydrated. Sections, 1 thick, and ultrathin sections from the same blocks were stained with 0.001 M Ruthenium Red. Semi-thin sections from cartilage fixed without heavy metal additives were, in addition, stained with the acidophilic fluorochrome Berberine sulphate. It was found that Ruthenium Red intensely stained the same pericellular zone that stained metachromatically with Toluidine Blue or fluoresced after staining with Berberine sulphate. Prior treatment with 0.05% cetylpyridinium chloride entirely blocked the three reactions. Previous digestion with 0.2 mg hyaluronidase/ml for 30 min at 37°C led to the abolition of the fluorescence reaction with Berberine sulphate. It is concluded that Ruthenium Red selectively stains cartilage acid glycosaminoglycans. With the electron microscope the pericellular zones were found to be built up of a three-dimensional branched meshwork of fibrils covered with a mantle of electron-dense material, presumably acid glycosaminoglycans bound to Ruthenium Red.