The movement and invasion of an insect baculovirus in tracheal cells of the armyworm, Pseudaletia unipuncta

The hypertrophy nuclear polyhedrosis virus of the armyworm, Pseudaletia unipuncta, causes a unique gradient of infected cells to form on the trachea. The movement and invasion of the virus apparently were not through adjacent intercellular membranes. The enveloped viruses emerged from the initially infected cell into an area between the cell plasma membrane and basal lamina, and then entered the uninfected tracheal cell either by lateral attachment and fusion of the viral envelope and the plasma membrane or by viropexis. The two methods of viral invasion into the cell suggest the presence of at least two phenotypically different enveloped viruses. Viropexis was initiated with an alignment of the peplomer spikes with regularly spaced, short radial striations on the inner coat of the plasma membrane. At a late state in viropexis, the viral envelope fused with the vacuole membrane, and an opening developed below the site of membrane fusion through which the nucleocapsid might enter the cytoplasm. Some nucleocapsids in membrane-lined vesicles resulting from viropexis appeared to be in a state of dissolution. Naked nucleocapsids were found along the nuclear envelope and within the nucleoplasm. No uncoating of the nucleocapsids was observed at the nucleopores, but uncoating seemed to occur in the nucleoplasm. Nucleocapsids were also found in the cytoplasm of nonsusceptible fat body cells, in which virus replication was not observed.     

The Nuclear Pore Complex and Nuclear Transport

Internal membrane bound structures sequester all genetic material in eukaryotic cells. The most prominent of these structures is the nucleus, which is bounded by a double membrane termed the nuclear envelope (NE). Though this NE separates the nucleoplasm and genetic material within the nucleus from the surrounding cytoplasm, it is studded throughout with portals called nuclear pore complexes (NPCs). The NPC is a highly selective, bidirectional transporter for a tremendous range of protein and ribonucleoprotein cargoes. All the while the NPC must prevent the passage of nonspecific macromolecules, yet allow the free diffusion of water, sugars, and ions. These many types of nuclear transport are regulated at multiple stages, and the NPC carries binding sites for many of the proteins that modulate and modify the cargoes as they pass across the NE. Assembly, maintenance, and repair of the NPC must somehow occur while maintaining the integrity of the NE. Finally, the NPC appears to be an anchor for localization of many nuclear processes, including gene activation and cell cycle regulation. All these requirements demonstrate the complex design of the NPC and the integral role it plays in key cellular processes.Taxonomically speaking, all life on earth falls into one of two fundamental groups, the prokaryotes and the eukaryotes. The prokaryotes, the first group to evolve, are single cell organisms bounded by a single membrane. About 1.5 billion years later, a series of evolutionary innovations led to the emergence of eukaryotes. Eukaryotes have multiple inner membrane structures that allow for compartmentalization within the cell, and therefore differentiation of the cell and regulation within it. Ultimately, the greater cellular complexity of eukaryotes allowed them to adopt a multicellular lifestyle, as seen in the plants, fungi and animals of today (reviewed in Field and Dacks 2009).Internal membrane bound structures sequester all genetic material in eukaryotic cells. The most prominent of these structures, which gives the eukaryotes their Greek-rooted name, is the nucleus—the central “kernel” (gr. “karyo-”) of the cell. The nucleus is bounded by a double membrane termed the nuclear envelope (NE), which separates the nucleoplasm and genetic material from the surrounding cytoplasm. However the genetic material in the nucleus is not totally isolated from the rest of the cell. Studded throughout the NE are portals called nuclear pore complexes (NPCs). The NPC is a highly selective, bidirectional transporter for a tremendous range of cargoes. Going into the nucleus, these cargoes include inner nuclear membrane proteins and all the proteins in the nucleoplasm. Going out are RNA-associated proteins that are assembled into ribosomal subunits or messenger ribonucleoproteins (mRNPs). Once transported, the NPC must ensure these cargos are retained in their respective nuclear and cytoplasmic compartments. All the while the NPC must prevent the passage of nonspecific macromolecules, yet allow the free diffusion of water, sugars, and ions. These many types of nuclear transport are regulated at multiple stages, providing a powerful extra level of cellular control that is not necessary in prokaryotes. Assembly, maintenance, and repair of the NPC must somehow occur while maintaining the integrity of the NE. Finally, the NPC appears to be an anchor for localization of many nuclear processes, including gene activation and cell cycle regulation (reviewed in Ahmed and Brickner 2007; Hetzer and Wente 2009). All these requirements demonstrate the complex design of the NPC and the integral role it plays in key cellular processes.     

STRUCTURAL FEATURES OF MESOSOMES (CHONDRIOIDS) OF BACILLUS SUBTILIS AFTER FREEZE-ETCHING

Freeze-etched cells of Bacillus subtilis have been studied with the electron microscope. The outer surface of the plasma membrane, i.e. the side facing the cell wall, is covered with numerous granules and short strands, each measuring approximately 50 A in diameter. These strands are occasionally seen to enter the cell wall. The inner surface of the plasma membrane, i.e. the side facing the cytoplasm, appears to be sparsely dotted with small particles measuring about 50 A. The envelope of mesosomes differs from the plasma membrane. Blunt protrusions arise from its outer surface; the inner surface appears smooth. Stalked particles, as described by other investigators after negative staining with phosphotungstic acid, were not observed on any membrane surface in our material. Preparations were also made of specimens prefixed in osmium tetroxide prior to freeze-etching. Under these conditions the bacterial membranes appeared to be surprisingly well preserved. In contrast to directly frozen, unfixed cells, some osmium tetroxide-fixed preparations showed a differentiation in cytoplasm and nucleoplasm, which made it possible to observe the close association of the mesosome with the latter.     

Nucleolus: the fascinating nuclear body

Nucleoli are the prominent contrasted structures of the cell nucleus. In the nucleolus, ribosomal RNAs are synthesized, processed and assembled with ribosomal proteins. RNA polymerase I synthesizes the ribosomal RNAs and this activity is cell cycle regulated. The nucleolus reveals the functional organization of the nucleus in which the compartmentation of the different steps of ribosome biogenesis is observed whereas the nucleolar machineries are in permanent exchange with the nucleoplasm and other nuclear bodies. After mitosis, nucleolar assembly is a time and space regulated process controlled by the cell cycle. In addition, by generating a large volume in the nucleus with apparently no RNA polymerase II activity, the nucleolus creates a domain of retention/sequestration of molecules normally active outside the nucleolus. Viruses interact with the nucleolus and recruit nucleolar proteins to facilitate virus replication. The nucleolus is also a sensor of stress due to the redistribution of the ribosomal proteins in the nucleoplasm by nucleolus disruption. The nucleolus plays several crucial functions in the nucleus: in addition to its function as ribosome factory of the cells it is a multifunctional nuclear domain, and nucleolar activity is linked with several pathologies. Perspectives on the evolution of this research area are proposed.     

Fine Structure of Strictly Anaerobic,Non-Spore-Forming,Gram-Negative Bacilli

The fine structure of a strain of Bacteroides insolitus has been studied by ultrathin sectioning and electron microscopy. Logarithmically growing cells were fixed both by osmic acid and potassium permanganate, and embedded in Epon. Thin sections were stained with uranyl acetate and examined. The periphery of the cell was composed of a wavy three-layered outer membrane (ca. 80 A), an intermediate layer (50–200 A), and three layered cytoplasmic membrane (ca. 80 A). Single or double bridges which connected the outer membrane with the cytoplasmic membrane were observed. Invagination of the cytoplasmic membrane was observed in no occasion. Independent, distinct, and uniform particles were not the main component of the cytoplasm. The cytoplasm was filled with more or less beaded reticulum-like structures. The nucleoplasm with fine fibrils was mainly dispersed continuously in rather regular cubic masses in an intermediate region between the center and the periphery of the cell. Contacts of the nucleoplasm with the cytoplasmic membrane were occasionally observed.     

Effects of Toluene and Phenethyl Alcohol on the Ultrastructure of Escherichia coli

Treatment of Escherichia coli cells with 0.25% toluene caused partial dissolution of the plasma membrane concomitant with displacement of nuclear material towards the cell periphery. By contrast, treatment with 0.25% phenethyl alcohol generated intracellular membranes.     

Protein diffusion in mammalian cell cytoplasm

We introduce a new method for mesoscopic modeling of protein diffusion in an entire cell. This method is based on the construction of a three-dimensional digital model cell from confocal microscopy data. The model cell is segmented into the cytoplasm, nucleus, plasma membrane, and nuclear envelope, in which environment protein motion is modeled by fully numerical mesoscopic methods. Finer cellular structures that cannot be resolved with the imaging technique, which significantly affect protein motion, are accounted for in this method by assigning an effective, position-dependent porosity to the cell. This porosity can also be determined by confocal microscopy using the equilibrium distribution of a non-binding fluorescent protein. Distinction can now be made within this method between diffusion in the liquid phase of the cell (cytosol/nucleosol) and the cytoplasm/nucleoplasm. Here we applied the method to analyze fluorescence recovery after photobleach (FRAP) experiments in which the diffusion coefficient of a freely-diffusing model protein was determined for two different cell lines, and to explain the clear difference typically observed between conventional FRAP results and those of fluorescence correlation spectroscopy (FCS). A large difference was found in the FRAP experiments between diffusion in the cytoplasm/nucleoplasm and in the cytosol/nucleosol, for all of which the diffusion coefficients were determined. The cytosol results were found to be in very good agreement with those by FCS.     

Diffusion-based transport of nascent ribosomes in the nucleus

Although the complex process of ribosome assembly in the nucleolus is beginning to be understood, little is known about how the ribosomal subunits move from the nucleolus to the nuclear membrane for transport to the cytoplasm. We show here that large ribosomal subunits move out from the nucleolus and into the nucleoplasm in all directions, with no evidence of concentrated movement along directed paths. Mobility was slowed compared with that expected in aqueous solution in a manner consistent with anomalous diffusion. Once nucleoplasmic, the subunits moved in the same random manner and also sometimes visited another nucleolus before leaving the nucleus.     

Vesicular stomatitis virus M protein in the nuclei of infected cells.

The M protein of vesicular stomatitis virus (VSV) was localized in the nuclei and cytoplasm of VSV-infected cells by subcellular fractionation and immunofluorescence microscopy. Nuclei isolated from VSV-infected Friend erythroleukemia cells were fractionated into a nuclear membrane and a nucleoplasm fraction by DNase digestion and differential centrifugation. G protein was present in the membrane fraction, and M protein was present in the nucleoplasm fraction. Immunofluorescence detection of M protein in the nucleus required that fixed cells be permeabilized with higher concentrations of detergent than were required for detection of M protein in the cytoplasm of VSV-infected BHK cells.     

THE NUCLEAR ENVELOPE : ITS STRUCTURE AND RELATION TO CYTOPLASMIC MEMBRANES

An electron microscope study of thin sections of interphase cells has revealed the following:— Circular pores are formed in the double nuclear envelope by continuities between the inner and outer membranes which permit contact between the nucleoplasm and the cytoplasm unmediated by a well defined membrane. The pores, seen in sections normal to the nuclear envelope, are profiles of the ring-shaped structures described by others and seen in tangential section. The inner and outer nuclear membranes are continuous with one another and enclose the perinuclear space. The pores contain a diffuse, faintly particulate material. A survey of cells of the rat derived from the embryonic ectoderm, mesoderm, and endoderm, and of a protozoan and an alga has revealed pores in all tissues examined, without exception. It is concluded that pores in the nuclear envelope are a fundamental feature of all resting cells. In certain cells, the outer nuclear membrane is continuous with membranes of the endoplasmic reticulum, hence the perinuclear space is continuous with cavities enclosed by those membranes. There are indications that this is true for all resting cells, at least in a transitory way. On the basis of these observations, the hypothesis is made that two pathways of exchange exist between the nucleus and the cytoplasm; by way of the perinuclear space and cavities of the endoplasmic reticulum and by way of the pores in the nuclear envelope.     

The nuclear envelope; its structure and relation to cytoplasmic membranes

An electron microscope study of thin sections of interphase cells has revealed the following:- Circular pores are formed in the double nuclear envelope by continuities between the inner and outer membranes which permit contact between the nucleoplasm and the cytoplasm unmediated by a well defined membrane. The pores, seen in sections normal to the nuclear envelope, are profiles of the ring-shaped structures described by others and seen in tangential section. The inner and outer nuclear membranes are continuous with one another and enclose the perinuclear space. The pores contain a diffuse, faintly particulate material. A survey of cells of the rat derived from the embryonic ectoderm, mesoderm, and endoderm, and of a protozoan and an alga has revealed pores in all tissues examined, without exception. It is concluded that pores in the nuclear envelope are a fundamental feature of all resting cells. In certain cells, the outer nuclear membrane is continuous with membranes of the endoplasmic reticulum, hence the perinuclear space is continuous with cavities enclosed by those membranes. There are indications that this is true for all resting cells, at least in a transitory way. On the basis of these observations, the hypothesis is made that two pathways of exchange exist between the nucleus and the cytoplasm; by way of the perinuclear space and cavities of the endoplasmic reticulum and by way of the pores in the nuclear envelope.     

The Relative Concentration of Solids in the Nucleolus, Nucleus, and Cytoplasm of the Developing Nerve Cell of the Chick

Growing and differentiating nerve cells of the fifth cranial ganglion of the chick embryo were studied by several means. During the period of 70 hours to 11 days of incubation (Hamburger-Hamilton stages 19 to 37) average cell mass increased more than 4.5 times while cells changed from relatively undifferentiated neuroblasts to morphologically characteristic nerve cells with long processes. By making simplifying assumptions about thickness of nucleus and nucleolus, relative to cytoplasmic thickness, it was possible to calculate solute concentration of nucleus and nucleolus relative to that of the cytoplasm from measurements of optical retardations through living cells. Differences in relative solute concentration were observed in nucleolus, cytoplasm, and nucleoplasm in the approximate ratio 1.2:1.0:0.8, respectively. The ratio remained essentially constant during the growth period examined despite the fact that the cell components grow at markedly different rates. This suggests that solid concentrations are physical characteristics of nucleus, nucleolus, and cytoplasm which are maintained even during rapid growth and differentiation. By cytochemical means it was demonstrated that mass increase in the nucleus is not associated with increase in deoxyribonucleic acid. Both ribonucleic acid and protein are in greater concentration in nucleolus and cytoplasm than in the nucleoplasm. Electron microscopy shows interruptions in the nuclear envelope as well as an approximately even distribution of electron density in nucleus and cytoplasm. It is pointed out that consistent differences in solid concentration can exist on either side of the nuclear envelope even though it contains "pores." Implications of these data are discussed.     

Cell age-related differences in the interaction of a potential-sensitive fluorescent dye with nuclear envelopes of Acetabularia mediterranea

Abstract. To study whether an electrical potential difference exists across the nuclear envelope or inner nuclear membrane of plant cells, the authors have used an optical probe of membrane potential, the cationic fluorescent dye, DiOC₆(3) (MW = 572.5). This dye was microinjected into the nucleoplasm of isolated Acetabularia nuclei (which are still surrounded by a thin layer of cytoplasm) and its subnuclear localization visualized by fluorescence microscopy. Striking differences, which seemed to be correlated with the developmental stage of the isolated nucleus, were observed. In nuclei isolated from cells at the stage of early cap stage formation, the dye was restricted to the nuclear envelope. In nuclei isolated from cells with intermediate or fully developed caps, there was increased nucleoplasmic staining, and the staining of the envelope was frequently diminished or abolished. In all nuclei, the dye remained within the nucleus after injection. Cytoplasmic staining was only observed when nuclei isolated from cells at the stage of early cap formation were incubated in a hyper- or hypo-tonic medium. Various ionophores, injected before the dye into the nucleoplasm, had no effect on the subsequent nuclear localization of DiOC₆(3), although they did rapidly induce nucleolar condensation in nuclei isolated from cells at the stage of early cap formation. The results suggested that the electrical properties of Acetabularia nuclear envelopes or inner nuclear membranes change during cell maturation. Furthermore, the retention of the dye in the nucleoplasm under isotonic conditions indicated that the nuclear pores were not open channels for molecules of this size.     

c-fos和c-myc在北方山溪鲵精子发生中的表达

用免疫组织化学方法检测原癌基因cf-os和c-myc蛋白在北方山溪鲵(Batrachuperus tibetanus)精子发生中的表达定位。结果显示,在精原细胞缓慢增殖期,8、9月,FOS阳性反应物出现在精原细胞的胞质及核膜外,10、11月,FOS在少量精原细胞的胞核中表达。在精原细胞快速增殖期,即翌年4月,FOS定位在精原细胞的胞质中;5月,FOS在大量的胞核中强阳性表达;6月,FOS定位于部分精母细胞核质和核膜下;7月,FOS在一些精子细胞的核质和核膜下表达。MYC在8、9月的部分精原细胞胞质中表达较弱,在101、1月阳性反应出现在个别精原细胞的核质中。翌年4月,MYC在精原细胞核周围的胞质中表达;5月在大量的精原细胞核膜下有强表达;6月,MYC在一些精母细胞核膜下表达;7月,MYC在部分精子细胞的核膜下弱表达。结果表明,北方山溪鲵的原癌基因cf-os和c-myc表达大强度在生精细胞发育中呈阶段性,表达的强度和细胞数量与细胞增殖的速度相一致。FOS和MYC在精原细胞内从胞质向胞核的转移与细胞快速增殖的时期相吻合。说明cf-os和c-myc对精原细胞有丝分裂有促进作用,并参与精母细胞成熟分裂的调控。     

External fibronectin of cultured human fibroblasts is predominantly a matrix protein

The distribution of a major glycoprotein (fibronectin) of human fibroblast cultures was studied in immunoelectron microscopy with peroxidase- or ferritin-labeled antibodies. External fibronectin was visualized in pericellular structures, in some areas on the growth substratum, and to a lesser degree in close association with the upper and lower surface membranes of the cell. The pericellular fibronectin-containing structures consisted of amorphous or vaguely fibrillar material forming strands or patches, 50-500 nm in diameter; the structures appeared to mediate distant cell-to-cell and cell-to-substrate contacts. When in close association with the plasma membrane, fibronectin markers were seen as discrete patches. The exact relationship between this form of fibronectin and the plasma membrane, however, remained open. Filamentous material was commonly seen in the cortical cytoplasm under patches of membrane-associated fibronectin. The distribution that we observed is consistent with the proposed roles of fibronectin in cell interactions with neighboring structures and with its presence in vivo as an extracellular glycoprotein in connective tissue matrix and basal laminae.     

Structure and expression of the maize (<Emphasis Type="Italic">Zea mays</Emphasis>L.) SUN-domain protein gene family: evidence for the existence of two divergent classes of SUN proteins in plants

Background  

The nuclear envelope that separates the contents of the nucleus from the cytoplasm provides a surface for chromatin attachment and organization of the cortical nucleoplasm. Proteins associated with it have been well characterized in many eukaryotes but not in plants. SUN (Sad1p/Unc-84) domain proteins reside in the inner nuclear membrane and function with other proteins to form a physical link between the nucleoskeleton and the cytoskeleton. These bridges transfer forces across the nuclear envelope and are increasingly recognized to play roles in nuclear positioning, nuclear migration, cell cycle-dependent breakdown and reformation of the nuclear envelope, telomere-led nuclear reorganization during meiosis, and karyogamy.     

Tubulin-specific antibody and the expression of microtubules in 3T3 cells after attachment to a substratum : Further evidence for the polar growth of cytoplasmic microtubules in vivo

Mouse 3T3 cells were allowed to attach to and spread on glass. The expression of cytoplasmic microtubules during the respreading process was monitored by immunofluorescence microscopy using monospecific antibody against tubulin. During radial attachment of the cells a ring of flattened cytoplasm is seen around the nucleus. Cytoplasmic microtubules then enter this spreading ring from the perinuclear region and elongate toward the plasma membrane. At later times microtubules appear perpendicular to the plasma membrane and seem to be in intimate contact with it giving the impression that they “stretch” the cytoplasm. When the cells assume their typical fibroblastic shape numerous microtubules are seen. They traverse the cytoplasm. Some come close to the plasma membrane and some bend to conform to the shape of the cell. Changes in microtubular organization correlate well with changes in cell shape. These results together with our previous observations on the assembly of cytoplasmic microtubules upon recovery from colcemid treatment suggest that microtubules may grow as polar structures from a microtubular organizing center towards the plasma membrane. The hypothesis that cytoplasmic microtubules might confer polarity on the cell is discussed.     

Secretion in dissociated human pulmonary mast cells. Evidence for solubilization of granule contents before discharge

Mast cells were enzymatically dissociated from human lung fragments that had been sensitized with serum from human allergic to ragweed and were enriched by isopyknic and velocity gradient sedimentation. Electron microscope examination showed that the mast cells were well preserved at the end of the dissociation and isolation and that the majority of their secretory granules contained crystalline structures. These structures exhibited three patterns--scrolls, gratings, and lattices--which all could be found in the same granule. The period of crystalline structures was found to be bimodal, with maxima at 150 and 75 A. Both periods were observed in gratings that had been tilted and in scrolls that had been cut obliquely, indicating that the various gross patterns are composed of the same basic substructure. After the mast cells were stimulated by rabbit anti-human IgE to release histamine, the contents of the granule were transformed from a crystalline to an amorphous state, and only granules with amorphous contents were seen discharging from the cell. Clusters of intermediate filaments were present around the granules with amorphous contents, both deep in the cytoplasm and discharging at the cell surface. Discharge occurred both by fusion of granule membranes with the plasma membrane and by fusion of granule membranes with other granule membranes that ultimately were continuous with the plasma membrane. After discharge, the granule residue was fibrillar. Cells challenged with anti-human IgE in calcium-free medium neither released histamine nor demonstrated morphologic changes in their granules. We conclude that the crystalline state represents a storage form for materials that are solubilized before fusion of the granule membrane with the plasma membrane and discharge.     

Structural components of the nuclear body in nuclei of Allium cepa cells

INTRODUCTIONSmall spherical nucleax bodies have long beenobserved in both hamal and plain interphasenuclei. In the case of animal cells, these nuclear bodies are generally called coiled bodies[1].As for plant cells, they have been vaxiously described as coiled bodies, ~somes, micronucleolior nucloolus-associated bodies because they sometimes appeared in the vicinity of nucleolusl2-4].Eaxly cytologists noted that nuclear bodies in platcells appeared as a tangle of coiled threads forming a …