Double Nuclear Membrane Research Articles

Assembly of the nuclear pore: biochemically distinct steps revealed with NEM, GTP gamma S, and BAPTA.

A key event in nuclear formation is the assembly of functional nuclear pores. We have used a nuclear reconstitution system derived from Xenopus eggs to examine the process of nuclear pore assembly in vitro. With this system, we have identified three reagents which interfere with nuclear pore assembly, NEM, GTP gamma S, and the Ca++ chelator, BAPTA. These reagents have allowed us to determine that the assembly of a nuclear pore requires the prior assembly of a double nuclear membrane. Inhibition of nuclear vesicle fusion by pretreatment of the membrane vesicle fraction with NEM blocks pore complex assembly. In contrast, NEM treatment of already fused double nuclear membranes does not block pore assembly. This indicates that NEM inhibits a single step in pore assembly--the initial fusion of vesicles required to form a double nuclear membrane. The presence of GTP gamma S blocks pore assembly at two distinct steps, first by preventing fusion between nuclear vesicles, and second by blocking a step in pore assembly that occurs on already fused double nuclear membranes. Interestingly, when the Ca2+ chelator BAPTA is added to a nuclear assembly reaction, it only transiently blocks nuclear vesicle fusion, but completely blocks nuclear pore assembly. This results in the formation of a nucleus surrounded by a double nuclear membrane, but devoid of nuclear pores. To order the positions at which GTP gamma S and BAPTA interfere with pore assembly, a novel anchored nuclear assembly assay was developed. This assay revealed that the BAPTA-sensitive step in pore assembly occurs after the second GTP gamma S-sensitive step. Thus, through use of an in vitro nuclear reconstitution system, it has been possible to biochemically define and order multiple steps in nuclear pore assembly.

Differential mitotic phosphorylation of proteins of the nuclear pore complex.

During each cell cycle, the nucleus of higher eukaryotes undergoes a dramatic assembly and disassembly. These events can be faithfully reproduced in vitro using cell-free extracts derived from Xenopus eggs. Such extracts contain three major N-acetylglucosaminylated proteins, p200, p97, and p60. All three become assembled into reconstituted nuclear pores. Here we show that p200, p97, and p60 exist in eggs in soluble high molecular mass complexes of 1000, 450, and 600 kDA, respectively. The bulk of p60 is stably associated with proteins of 58 and 54 kDa, while p200 is associated with a fraction of p60 in a separate complex lacking p58 and p54. Upon examining the behavior of these proteins in the cell cycle, we find that p200 and p97 are highly phosphorylated at mitosis, both in vivo and in vitro. Moreover, in extracts that cycle between interphase and mitosis, p200 and p97 are specifically phosphorylated at mitosis. Corresponding with their mitotic phosphorylation, both p200 and p97 are specific substrates for purified mitotic Cdc2 kinase, whereas nucleoporin p60 is not. Analysis indicates that the size of the complexes containing the pore N-acetylglucosamine glycoproteins does not change during mitosis, suggesting that such complexes represent stable multicomponent modules into which the nucleus disassembles at mitosis.

Phospholipids in the nucleus—metabolism and possible functions

Most of the phospholipids in the nuclear envelope are contained in the double nuclear membrane, and this has an active lipid metabolism consistent with its origins as a component of the endoplasmic reticular system. However, even after removal of the nuclear membrane with detergents, some phospholipids, mostly of unknown location and function, remain. Amongst these are all of the components of what appears to be a nuclear polyphosphoinositide signalling system, distinct from the well-established inositide pathway found in the plasma membrane. The consequences for nuclear function of the activation of these two inositide pathways are discussed, with a detailed consideration of proposed intranuclear functions for protein kinase C, and the maintenance of nuclear Ca 2+ homoeostasis.

A role for ADP-ribosylation factor in nuclear vesicle dynamics.

Two distinct steps in nuclear envelope assembly can be assayed in vitro: the protein-mediated binding of nuclear-specific vesicles to chromatin, and the subsequent fusion of these vesicles to enclose the chromatin within a double nuclear membrane. Nuclear vesicle fusion, like fusion in the secretory pathway, requires ATP and cytosol and is inhibited by nonhydrolysable GTP analogues. The sensitivity of nuclear vesicle fusion to GTP-gamma S requires a GTP-dependent soluble factor, the properties of which are strikingly similar to a GTP-dependent Golgi binding factor (GGBF) that inhibits Golgi vesicle fusion in the presence of GTP-gamma S and belongs to the ADP-ribosylation factor (ARF) family of small GTPases. In the presence of GTP-gamma S, ARF proteins and alpha-, beta-, gamma-, delta-COP ('coatomer') subunits are associated with Golgi transport vesicles, but the exact roles of ARF proteins in secretion are not yet understood. We report here that purified ARF1 and GGBF have GTP-dependent soluble factor activity in the nuclear vesicle fusion assay. Our results show that the function of ARF is not limited to the Golgi apparatus, and indicate that there may be a link between the formation of nuclear vesicles during mitosis and proteins involved in secretion.

Ultrastructure of the freeze-etched spore of Ascosphaera apis, an entomopathogenic fungus of the honeybee Apis mellifera

The freeze-fractured outer surface of the spore wall of Ascophaera apis is covered with granules which are 21.5 nm in diameter. The cross-fractured spore wall consists of subunits which are 9 nm in diameter. The inner surface of the spore wall is granular and has many stud-like projections measuring approximately 120 nm long × 26 nm wide. The convex face of the spore membrane carries many particles as well as many depressions which are complementary to the projections on the inner surface of the spore wall. The nucleus possesses double nuclear membranes which contain numerous well-defined nuclear pores. In the cytoplasm of the spore there are many mitochondria which have well-defined cristae and many particles on the internal membranes. The sporoplasm contains many lipid droplets which possess concentric smooth-surfaced lamellae.

Spontaneous formation of nucleus-like structures around bacteriophage DNA microinjected into Xenopus eggs.

We have found that injection of bacteriophage lambda DNA into unfertilized Xenopus eggs causes the assembly around the DNA of structures resembling typical eucaryotic cell nuclei. These spherical structures begin to form 60-90 min after injection. They contain lambda DNA and are bounded by a phase-dense envelope. Immunofluorescent staining of the lambda-DNA-containing structures with anti-lamin antibody reveals the presence of the lamin nuclear proteins at the periphery of the structure, a pattern identical to that of embryonic nuclei. Electron microscopy reveals that the injected DNA is surrounded by a double bilayer nuclear membrane containing nuclear pore complexes. The "nuclei" containing lambda DNA respond to modulators of the Xenopus cell cycle in a manner that mimics the response of embryonic nuclei to these modulators during mitosis. These results suggest that nuclear reassembly and breakdown occur independently of specific DNA sequence information.

An ultrastructural examination of developing and mature euspermatozoa in pyrazus ebeninus (Mollusca, Gastropoda, Potamididae)

The mesogastropod Pyrazus ebeninus, produces true spermatozoa (here termed euspermatozoa) and multi-flagellate, mobile cells (here termed paraspermatozoa). The mature paraspermatozoon consists of an elongateconical ‘head’ (6.5–8.5 μm in length), constructed of an electron-dense mosaic sheath surrounding a similarly dense, rod-shaped nuclear core (which runs almost the full length of the head). An acrosome-like structure forms the apex of the head. Five to eight axonemes are fixed to the posterior extremity of the nuclear core, each by means of an attachment complex (dense attachment rod, centriolar cap and centriole). A short (3–4 μm) ‘midpiece’ zone follows the head and consists of the multiple axonemes interspersed with very elongate mitochondria. A tuft of short (20 μm) tails (termed minor tails) emerges from the midpiece in addition to one very long tail (termed the major tail) ensheathed in dense granules which resemble glycogen granules. A single membrane surrounds head, midpiece and tails whilst the nuclear core retains the original double nuclear membrane. Developmentally, the multiple axonemes arise from one of a pair of wheel-shaped arrangements of centrioles and attach to posterior indentations in the nucleus prior to its transformation into the nuclear core. Dense vesicles, derived apparently from the endoplasmic reticulum, accumulate along and around the developing nuclear core and (in the presence of microtubules) condense into the mosaic head sheath. Cytoplasmic mitochondria elongate and collect at the posterior axis of the cell, where, together with the axonemes, they form the midpiece. Features not previously reported in other ultrastructural studies of paraspermatozoa include the acrosome-like structure of the head, the structure of the midpiece zone, the glycogen sheath of the major tail, the dense annular structure at the junction of the midpiece and major tail and the presence of microtubules in the final phase of head and midpiece maturation. Some features of the euspermatozoon are also described and the comparative ultrastructure of mature and developing paraspermatozoa and their possible functions in the Gastropoda, are reviewed.

Freeze-etching observations of trophozoites of pathogenic Entamoeba histolytica

Pathogenic Entamoeba histolytica trophozoites were studied by the freeze-etching (FE) technique of electron microscopy. Surface replicas of intact cell membranes were highly convoluted with numerous invaginations, evaginations, and undulations. Sperical depressions and elevations varying from 0.5 mu to 1.0 mu in diameter were commonly present on the external cell membrane and appeared to represent an extracellular secretory mechanism of trophozoites. Cleaved surfaces of amebae exhibited a granular and lumpy cytoplasm in which there were many vesicles and vacuoles that ranged in diameter from 0.2 mu to 9.0 mu. Some vacuoles contained tightly enveloped bacteria, while others contained bacteria and host cytocomponents. Occasional vesicles and vacuoles appeared to be fused to each other. Replicas of FE nucleus were enclosed by double nuclear membranes which were fenestrated by numerous sperical pores measuring approximately 640 A in diameter and spaced at intervals of 650 A. Counts of nuclear pores were possible and indicated 35 pores per square micron on the nuclear envelope. Golgi apparatus, mitochondria and well formed endoplasmic reticulum were absent in FE replicas. This was in agreement with electron microscope observations on thin sections previously reported by other investigators.

Some electron microscopic studies on intact nuclear 'ghosts' and nuclear membrane fragments

The electron optical images of negatively stained intact nuclear ‘ghosts’ are readily identified as flattened and sometimes folded bodies, not unlike those given by mammalian erythrocyte ‘ghosts’. Even at low magnifications ( x 5000) the nuclear pore complexes are clearly revealed and can therefore be used as morphological markers for nuclear membrane. At higher magnifications the nuclear pore complexes are seen to be composed of an eight-sided annulus surrounding a central granule, an inner ring of material, and possibly radial fibrils. Very often fine detail appears to be obscured owing to the fact that there are two layers of double nuclear membrane lying flat on the carbon support film. Occasionally the nuclear ‘ghosts’ appear to be torn apart, in all probability because of the spreading and surface-tension forces applied during the preparation of the negatively stained specimens. In this instance the nuclear pore complexes usually remain intact and the surrounding membrane is disrupted. The nuclear pore complexes are spread as a single layer and greater detail is revealed within the annuli. This is particularly so when partially disrupted nuclear pore complexes are studied. The octagonal annulus appears to be composed of circular macromolecules approximately 20 nm in external diameter with a 5 nm diameter central hole. These macromolecules are linked together and partly masked by other diffuse material. It is proposed that one or more of these macromolecules underlies each of the eight annular subunits. A model for the nuclear pore complex is presented and compared with those proposed by other authors.

Fine Structures Of Trichomonas Tenax And Trichomonas Hominis

Trichomonas tenax(T. tenax) and Trichomonas hominis (T. hominis) were collected, cultured and sampled for comparative microscopical studies using electron microscope. 1. Both flagellates were oval in shape and surrounded by a distinct outer membrane. Five recurrent flagella and one anterior flagellum had, each, 9 paris of peripheral and 1 pair of central fibrils, Undulating membrane was curved over the recurrent flagella, and bended in the middle at right angles with cell surface. Cytostome, engulfing bacteria, was observed in T. hominis. 2. In the cytoplasm, there were fine dense glycogen particles, and vacuoles containing ingested materials. Dense pigment rods were also observed in both flagellates, but the rods were not distributed around the vacuoles in T. hominis. 3. In T. tenax axostyle appeared as a cup-shaped structure comprising a single row of 41 fibrils, each about 120 a in diameter. It enclosed glycogen particles, and the open side was faced to the nucleus. 4. Endoplasmic reticulum was observed around the nucleus, but it was less developed in T. hominis. 5. Nucleus was ovoid having double nuclear membrane, which was clearly defined in T. hominis. 6. Blepharoplast, parabasal body, Golgi appartus and mitochondrion was not observed in both flagellates.

The preparation of nucleated erythrocyte ghosts from avian erythrocytes.

Synopsis A method is described for the preparation of erythrocyte ghosts, which are haemoglobin‐depleted, from domestic fowl and turkey erythrocytes. These erythrocyte ghosts retain their ellipsoid shape and their nuclei, which are shown to possess the typical double nuclear membrane. In the cytoplasmic compartment of the ghosts, mitochondria have been clearly observed. There is little or no indication of endoplasmic reticulum. Filamentous structures have been seen adhering to the outer nuclear membrane, the mitochondria and the inner surface of the plasma membrane. These filaments may extend between the plasma membrane and the surfaces of the nucleus and mitochondria, thereby providing a form of structural anchorage for these organelles. It is a consistent observation that the nucleus of the intact erythrocyte and its ghost always occupies a central position, so some structure must be holding the nucleus otherwise one would expect to occasionally find it positioned off‐centre. Significantly, very few of ...

Chemical composition of nuclear crystals in leaves ofStellaria media L.

The chemical composition of crystalline inclusions, which have been detected in the double nuclear membrane with the electron microscope (Perrin 1969) was studied by enzymatic digestion. The crystals were digested in thin sections by pepsin and after 1 hour of incubation, nearly all the crystalline inclusions had disappeared entirely leaving empty spaces.

Studies on the fine structure of the red alga Porphyridium cruentum

In a comparative study of the suitability of OsO4, KMnO4, and acrolein as fixatives for an electron microscopical examination of the fine structure of the unicellular red alga Porphyridium cruentum, it was found that the use of 10% acrolein in artificial sea water with postfixation in 1% OsO4 produced the best overall results. In such preparations the presence of mitochondria, dictyosomes, double nuclear membrane with pores, endoplasmic reticulum, and a chloroplast with finger-like lobes composed of parallel lamellae of single disks were clearly evident.

Electron Microscopy of Astasia longa*

SYNOPSIS. Cells of Astasia longa (Jahn) were grown on a simple defined medium, fixed in the stationary phase of growth with OsO4, stained with uranyl acetate, and embedded in Vestopal for electron microscopy. The cell wall was found to be a membranous structure 100 Å thick. A double nuclear membrane, nucleolus, and many darkly staining bodies were observed in the nucleus. Cells contained ribosomes and elaborate Golgi bodies, but no well developed endoplasmic reticulum. The numerous mitochondria typically had well‐defined cristae running longitudinally in the mitochondrion. Two bodies of unknown function were observed, one sometimes seen to surround mitochondria, the other bounded by a double membrane. A model of the reservoir structure of the euglenoids is presented, based on observations of the reservoir of A. longa and on published data from Euglena gracilis and E. viridis.

The deep invagination of the inner nuclear membrane into the nucleoplasm in the ascites hepatoma cells

IT has b een well established that the nuclear envelope of the interphase cell, in animals and in some kinds of plants, is composed of two parallel membranes [l, 3, 121. Some morphological evidences suggesting the nuclear-cytoplasmic interrelationships have also been demonstrated in electron micrographs: the existence of continuities between the membranes of the nuclear envelope and those of the endoplasmic reticulum [4, 12, 131; of several types of discontinuities in the nuclear envelope 15, 131; of deep invaginations of double nuclear membranes into the nuclei, engulfing a portion of cytoplasmic materials [2, 71; of protrusions of the outer nuclear membrane [7, 121; of “bleb” formations of nuclei towards the cytoplasm [6, 91; and of annulate’lamellae [lo, 111. The interpretation that the nuclear membrane is another local differentiation of the endoplasmic reticulum was proposed by Watson [la, 131 and supported by Palade [8]. In the course of electron microscopic study of various ascites hepatomas of rats, the author has met the picture showing a new type of the invagination of nuclear membrane into the nucleoplasm, which has not hitherto been recorded. The present

The origin and fate of the nuclear membrane in meiosis.

The behaviour of the nuclear membrane during meiotic division in locust spermatocytes has been studied by electron microscopy. Preliminary observations were made on living cells from locusts, grasshoppers and beetles by phase-contrast microscopy and on snail and newt spermatocytes by electron microscopy. The mitochondria come into close association with the nuclear membrane during prophase and are often clustered round regions of degenerating nuclear membrane. The mitochondrial membranes sometimes appear to be fused with the nuclear membrane, and gaps in the latter are often found near mitochondria. Lamellar stacks are found in the cytoplasm during early prophase and closely resemble the nuclear membrane in the same cells ; they are generally annulate in spermatogonia and primary spermatocytes butsmooth-walled in secondary spermatocytes. There is evidence that the stacks arise by repeated folding of the nuclear membrane and become converted into endoplasmic reticulum. After division, the daughter cell chromosomes are at first devoid of a surrounding membrane. Elements of the endoplasmic reticulum accumulate between the mitochondria and gradually surround the chromosomes. These elements fuse to form a continuous double nuclear membrane. It is suggested that the nuclear membrane, endoplasmic reticulum and cell membrane are composed of the same material, which can be transformed into different structures according to the needs of the cell. The nuclear membrane is converted into endoplasmic reticulum during division and stored in the cytoplasm . The new nuclear membranes in the daughter cells are formed by the fusion of elements of the endoplasmic reticulum.

Electron microscopic investigations on the leptomonad form of Leishmania donovani

1. 1. The leptomonad form of the protozoon, Leishmania donovani, has been studied electron-microscopically after ultrathin sectioning, as well as after treatment with distilled water following fixation. 2. 2. The pellicle has been found to be formed of two layers: an outer thin continuous layer and an inner thicker layer formed of parallel fibrils. 3. 3. The nucleus is provided with a double nuclear membrane and inside contains a number of distinct structures, which have been interpreted here as the chromosomes. 4. 4. The mitochondria show the typical structure with a distinct membrane enclosing a system of microvilli. 5. 5. The flagellum consists of ten fibrils running longitudinally along its length. Each of these fibrils is probably formed of two subfibrils. 6. 6. The flagellar fibrils terminate internally in granules which lie near the parabasal body. The latter is a transversally elongated body enclosed in a membrane.

The fine structure of the cells in mouse sarcoma 37 ascitic fluids.

The tumour cells and the reaction cells in Sarcoma 37 ascitic fluids have been studied in thin sections with the electron microscope. The reaction cells were either leucocytes or much larger acidophilic peritoneal cells of the same dimensions as the tumour cells; the peritoneal cells formed as much as 20 per cent of the large cell population. The fine structure of the cells is described and some new observations recorded. It has been found that the cell membrane of eosinophil granulocytes has a laminated composition and the characteristic granules of these cells a double limiting membrane. The pores in the double nuclear membrane of the peritoneal cells have been observed to have a fine line running across them. In the tumour cells, a rounded granular body with a central dense area has been found in the region of the centrosome; these cells were also seen to contain rows of parallel smooth surfaced cisternae lying 150 mmicro apart similar to those hitherto only observed in spermatids. There was a feltwork of fine filaments in the cytoplasm of the centrosome region of the tumour cells. The cytoplasmic fine structure underlying the basophilia of the tumour cells and the acidophilia of the peritoneal cells is compared and discussed.