Endoplasmic reticulum targeted GFP reveals ER organization in tobacco NT-1 cells during cell division.

The endoplasmic reticulum (ER) of plant cells undergoes a drastic reorganization during cell division. In tobacco NT-1 cells that stably express a GFP construct targeted to the ER, we have mapped the reorganization of ER that occurs during mitosis and cytokinesis with confocal laser scanning microscopy. During division, the ER and nuclear envelope do not vesiculate. Instead, tubules of ER accumulate around the chromosomes after the nuclear envelope breaks down, with these tubules aligning parallel to the microtubules of the mitotic spindle. In cytokinesis, the phragmoplast is particularly rich in ER, and the transnuclear channels and invaginations present in many interphase cells appear to develop from ER tubules trapped in the developing phragmoplast. Drug studies, using oryzalin and latrunculin to disrupt the microtubules and actin microfilaments, respectively, demonstrate that during division, the arrangement of ER is controlled by microtubules and not by actin, which is the reverse of the situation in interphase cells.     

Role of the nuclear envelope in synthesis, processing, and transport of membrane glycoproteins

The outer nuclear membrane is morphologically similar to rough endoplasmic reticulum. The presence of ribosomes bound to its cytoplasmic surface suggests that it could be a site of synthesis of membrane glycoproteins. We have examined the biogenesis of the vesicular stomatitis virus G protein in the nuclear envelope as a model for the biogenesis of membrane glycoproteins. G protein was present in nuclear membranes of infected Friend erythroleukemia cells immediately following synthesis and was transported out of nuclear membranes to cytoplasmic membranes with a time course similar to transport from rough endoplasmic reticulum (t 1/2 = 5-7 min). Temperature-sensitive mutations in viral membrane proteins which block transport of G protein from endoplasmic reticulum also blocked transport of G protein from the nuclear envelope. Friend erythroleukemia cells and NIH 3T3 cells differed in the fraction of newly synthesized G protein found in nuclear membranes, apparently reflecting the relative amount of nuclear membrane compared to endoplasmic reticulum available for glycoprotein synthesis. Nuclear membranes from erythroleukemia cells appeared to have the enzymatic activities necessary for cleavage of the signal sequence and core glycosylation of newly synthesized G protein. Signal peptidase activity was detected by the ability of detergent-solubilized membranes of isolated nuclei to correctly remove the signal sequence of human preplacental lactogen. RNA isolated from the nuclear envelope was highly enriched for G protein mRNA, suggesting that G protein was synthesized on the outer nuclear membrane rather than redistributing to nuclear membranes from endoplasmic reticulum before or during cell fractionation. These results suggest a mechanism for incorporation of membrane glycoproteins into the nuclear envelope and suggest that in some cell types the nuclear envelope is a major source of newly synthesized membrane glycoproteins.     

Actin-filled nuclear invaginations indicate degree of cell de-differentiation

For years the existence of nuclear actin has been heavily debated, but recent data have clearly demonstrated that actin, as well as actin-binding proteins (ABPs), are located in the nucleus. We examined live EGFP-actin-expressing cells using confocal microscopy and saw the presence of structures strongly resembling actin filaments in the nuclei of MDA-MB-231 human mammary epithelial tumor cells. Many nuclei had more than one of these filamentous structures, some of which appeared to cross the entire nucleus. Extensive analysis, including fluorescence recovery after photobleaching (FRAP), showed that all EGFP-actin in the nucleus is monomeric (G-actin) rather than filamentous (F-actin) and that the apparent filaments seen in the nucleus are invaginations of cytoplasmic monomeric actin. Immunolocalization of nuclear pore complex proteins shows that similar invaginations are seen in cells that are not overexpressing EGFP-actin. To determine whether there is a correlation between increased levels of invagination in the cell nuclei and the state of de-differentiation of the cell, we examined a variety of cell types, including live Xenopus embryonic cells. Cells that were highly de-differentiated, or cancerous, had an increased incidence of invagination, while cells that were differentiated had few nuclear invaginations. The nuclei of embryonic cells that were not yet differentiated underwent multiple shape changes throughout interphase, and demonstrated numerous transient invaginations of varying sizes and shapes. Although the function of these actin-filled invaginations remains speculative, their presence correlates with cells that have increased levels of nuclear activity.     

The mechanism of cytoplasmic streaming in characean algal cells: sliding of endoplasmic reticulum along actin filaments

Electron microscopy of directly frozen giant cells of characean algae shows a continuous, tridimensional network of anastomosing tubes and cisternae of rough endoplasmic reticulum which pervade the streaming region of their cytoplasm. Portions of this endoplasmic reticulum contact the parallel bundles of actin filaments at the interface with the stationary cortical cytoplasm. Mitochondria, glycosomes, and other small cytoplasmic organelles enmeshed in the endoplasmic reticulum network display Brownian motion while streaming. The binding and sliding of endoplasmic reticulum membranes along actin cables can also be directly visualized after the cytoplasm of these cells is dissociated in a buffer containing ATP. The shear forces produced at the interface with the dissociated actin cables move large aggregates of endoplasmic reticulum and other organelles. The combination of fast-freezing electron microscopy and video microscopy of living cells and dissociated cytoplasm demonstrates that the cytoplasmic streaming depends on endoplasmic reticulum membranes sliding along the stationary actin cables. Thus, the continuous network of endoplasmic reticulum provides a means of exerting motive forces on cytoplasm deep inside the cell distant from the cortical actin cables where the motive force is generated.     

Interphase Nuclei of Many Mammalian Cell Types Contain Deep, Dynamic, Tubular Membrane-bound Invaginations of the Nuclear Envelope

The nuclear envelope consists of a doublemembraned extension of the rough endoplasmic reticulum. In this report we describe long, dynamic tubular channels, derived from the nuclear envelope, that extend deep into the nucleoplasm. These channels show cell-type specific morphologies ranging from single short stubs to multiple, complex, branched structures. Some channels transect the nucleus entirely, opening at two separate points on the nuclear surface, while others terminate at or close to nucleoli. These channels are distinct from other topological features of the nuclear envelope, such as lobes or folds.

The channel wall consists of two membranes continuous with the nuclear envelope, studded with features indistinguishable from nuclear pore complexes, and decorated on the nucleoplasmic surface with lamins. The enclosed core is continuous with the cytoplasm, and the lumenal space between the membranes contains soluble ER-resident proteins (protein disulphide isomerase and glucose-6-phosphatase).

Nuclear channels are also found in live cells labeled with the lipophilic dye DiOC₆. Time-lapse imaging of DiOC₆-labeled cells shows that the channels undergo changes in morphology and spatial distribution within the interphase nucleus on a timescale of minutes.

The presence of a cytoplasmic core and nuclear pore complexes in the channel walls suggests a possible role for these structures in nucleo–cytoplasmic transport. The clear association of a subset of these structures with nucleoli would also be consistent with such a transport role.

     

Nucleus-associated actin in Amoeba proteus

The presence, spatial distribution and forms of intranuclear and nucleus-associated cytoplasmic actin were studied in Amoeba proteus with immunocytochemical approaches. Labeling with different anti-actin antibodies and staining with TRITC-phalloidin and fluorescent deoxyribonuclease I were used. We showed that actin is abundant within the nucleus as well as in the cytoplasm of A. proteus cells. According to DNase I experiments, the predominant form of intranuclear actin is G-actin which is associated with chromatin strands. Besides, unpolymerized actin was shown to participate in organization of a prominent actin layer adjacent to the outer surface of nuclear envelope. No significant amount of F-actin was found in the nucleus. At the same time, the amoeba nucleus is enclosed in a basket-like structure formed by circumnuclear actin filaments and bundles connected with global cytoplasmic actin cytoskeleton. A supposed architectural function of actin filaments was studied by treatment with actin-depolymerizing agent latrunculin A. It disassembled the circumnuclear actin system, but did not affect the intranuclear chromatin structure. The results obtained for amoeba cells support the modern concept that actin is involved in fundamental nuclear processes that have evolved in the cells of multicellular organisms.     

The nuclear reticulum in placental cells ofLilium regale is a part of the endomembrane system

Summary Placental cells line the ovarian transmitting tract inLilium regale and produce exudates for secretion. Sections through the highly lobed nuclei of these cells reveal the presence of membrane profiles which form vesicles with varying dimensions in cross section. Computer reconstruction of the nucleus reveals that the vesicle profiles form a complex reticulum of tubular cisternae, which spans the whole nucleus, enclosing a maze of continuous lumen space. Connections between the vesicles and the inner nuclear envelope are visible at various points along the nuclear envelope. This complex network of tubules which constitutes the reticulum arises from the inner nuclear membrane. The nuclear reticulum dramatically increases the inner-envelope surface area, comprising 82% of the total membrane perimeter of inner nuclear envelope and nuclear reticulum. The inner nuclear envelope invaginates into the nucleus forming the nuclear reticulum and the outer nuclear envelope evaginates into the endoplasmic reticulum (ER), indicating that there is a continuity between the lumens of the nuclear reticulum and the ER. The nuclear reticulum is labelled with zinc iodide-osmium tetroxide, a staining pattern identical to that seen in the ER. Positive reaction to the zinc iodide-osmium tetroxide indicates that the nuclear reticulum is a site for Ca²⁺ deposition. The nuclear reticulum forms an extension of the endomembrane system which reaches deep into the nucleoplasm. The lumenal continuity of this system means that there is a channel for communication from the cytoplasm into the nucleoplasm, and that this channel sequesters calcium.Abbreviations ER endoplasmic reticulum - TEM transmission electron microscope - ZIO zinc iodide-osmium tetroxide     

Tissue slices from living root caps as a model system in which to study cytodifferentiation of polar cells

Tissue slices of living root caps of cress (Lepidium sativum L.), two to three cell layers in thickness, were prepared by a microsurgical procedure. The viability, cellular structures and cytoplasmic movement of the cells were examined in the light microscope. Nuclei, amyloplasts, vacuoles and endoplasmic reticulum were identified and their positions confirmed after fixation and observation of the same cells in the electron microscope. The distribution of microtubules was shown by immunocytochemistry. During germination, microtubules appear first at the distal edges of the statocytes, while in mature statocytes a distal domain of criss-crossed microtubules could be distinguished from a proximal domain with transversally oriented microtubules. Microfilaments in young statocytes form a nuclear enclosure; in mature statocytes bundles of microfilaments fan out into the cell cortex. The transition from statocytes to secretion cells is accompanied by a more pronounced cortical network of microfilaments, while the nucleus-associated microfilaments remain visible. It is suggested that these microfilaments play a role in the positioning of the nucleus and the translocation of endoplasmic reticulum.Abbreviations ER endoplasmic reticulum - MF microfilament - MT microtubule     

Tau‐induced nuclear envelope invagination causes a toxic accumulation of mRNA in Drosophila

The nucleus is a spherical dual‐membrane bound organelle that encapsulates genomic DNA. In eukaryotes, messenger RNAs (mRNA) are transcribed in the nucleus and transported through nuclear pores into the cytoplasm for translation into protein. In certain cell types and pathological conditions, nuclei harbor tubular invaginations of the nuclear envelope known as the “nucleoplasmic reticulum.” Nucleoplasmic reticulum expansion has recently been established as a mediator of neurodegeneration in tauopathies, including Alzheimer's disease. While the presence of pore‐lined, cytoplasm‐filled, nuclear envelope invaginations has been proposed to facilitate the rapid export of RNAs from the nucleus to the cytoplasm, the functional significance of nuclear envelope invaginations in regard to RNA export in any disorder is currently unknown . Here, we report that polyadenylated RNAs accumulate within and adjacent to tau‐induced nuclear envelope invaginations in a Drosophila model of tauopathy. Genetic or pharmacologic inhibition of RNA export machinery reduces accumulation of polyadenylated RNA within and adjacent to nuclear envelope invaginations and reduces tau‐induced neuronal death. These data are the first to point toward a possible role for RNA export through nuclear envelope invaginations in the pathogenesis of a neurodegenerative disorder and suggest that nucleocytoplasmic transport machinery may serve as a possible novel class of therapeutic targets for the treatment of tauopathies.     

The role of the nuclear pore complex in adenovirus DNA entry.

Adenovirus targets its genome to the cell nucleus by a multistep process involving endocytosis, membrane penetration and cytoplasmic transport, and finally imports its DNA into the nucleus. Using an immunochemical and biochemical approach combined with inhibitors of nuclear import, we demonstrate that incoming viral DNA and DNA-associated protein VII enter the nucleus via nuclear pore complexes (NPCs). Depletion of calcium from nuclear envelope and endoplasmic reticulum cisternae by ionophores or thapsigargin blocked DNA and protein VII import into the nucleus, but had no effect on virus targeting to NPCs. Calcium-depleted cells were capable of disassembling incoming virus. In contrast, inhibitors of cytosolic O-linked glycoproteins of the NPC blocked virus attachment to the nuclear envelope, capsid disassembly and also nuclear import of protein VII. The data indicate that NPCs have multiple roles in adenovirus entry into cells: they contain a virus-binding and/or dissociation activity and provide a gateway for the incoming DNA genome into the nucleus.     

DIVISION IN THE DINOFLAGELLATE GYRODINIUM COHNII (SCHILLER) : A New Type of Nuclear Reproduction

Dinoflagellates are of interest because their chromosomes resemble the nucleoplasm of prokaryotes both chemically and ultrastructurally. We have studied nuclear division in the dinoflagellate Gyrodinium cohnii (Schiller), using cells obtained from cultures undergoing phasic growth. Electron micrographs of serial sections were used to prepare three-dimensional reconstructions of nuclei and chromosomes at various stages of nuclear division. During division, a complex process of invagination of the intact nuclear envelope takes place at one side of the nucleus and results in the formation of parallel cylindrical cytoplasmic channels through the nucleus. These invaginations contain bundles of microtubules, and each of the bundles comes to lie in the cytoplasm of a cylindrical channel. Nuclear constriction occurs perpendicular to these channels without displacement of the microtubules. There are no associations between chromosomes and the cytoplasmic microtubules. In dividing cells most chromosomes become V-shaped, and the apices of the V's make contact with the membrane surrounding cytoplasmic channels. It is proposed that the membrane surrounding cytoplasmic channels in the dividing nucleus may be involved in the separation of daughter chromosomes. Thus, dinoflagellates may resemble prokaryotes in the manner of genophore separation as well as in genophore chemistry and ultrastructure.     

3T3 cells have nuclear invaginations containing F-actin

Nuclear envelope invaginations occur in many kinds of cell. Double-labeling of 3T3 cells with Hoechst 33342 strain for DNA and phalloidin-rhodamine for F-actin, show that some nuclei appear to contain tangled knots of F-actin. Concanavalin A-fluorescein staining for membranes shows that the knots are continuations of the nuclear envelope. Although they contain F-actin, the knots appear by electron microscopy to be cytoplasmic invaginations lacking microfilaments. Since we have shown previously that nuclear-membrane associated actin forms perinuclear shells in 3T3 cells, we propose that nuclear knots also are composed of actin associated with the nuclear membrane. 3T3 nuclei also contain nuclear invaginations of a second kind. These invaginations lie perpendicular to the first type and lack F-actin.     

Stable expression and visualization of Mat-8 (FXYD-3) tagged with a fluorescent protein in Chinese Hamster Ovary (CHO)-K1 cells

A type I transmembrane protein, Mat-8 (FXYD-3), was tagged with fluorescent protein, Discosoma red fluorescent protein, at the carboxyl terminal cytoplasmic tail, and stably expressed in Chinese Hamster Ovary (CHO)-K1 cells. The fluorescence signal was distributed in intracellular membranes, being not only detected around the nuclear envelope but also partly overlapping with an endoplasmic reticulum marker. Subcellular fractionation by density gradient centrifugation supported this partial overlapping. The spherical structures observed were not colocalized with markers for lysosomes, endosomes, and Golgi bodies, suggesting that Mat-8 is distributed in a distinct endoplasmic reticulum region and the nuclear envelope after synthesis on membrane-bound ribosomes.Revisions requested 15 April 2005; Revisions received 11 May 2005     

Comparative scanning and transmission electron microscopy studies of the ependyma of the central canal in the spinal cord of primates. I. Electron optical image of the ependyma in the central canal of the spinal cord of the callithrix monkey (Callithrix jacchus, Linné 1758)

The ependyma lining the central canal of the spinal cord of adult males and females monkey, Callithrix jacchus, was examined by scanning and transmission electron microscopy. The cross section of the lumen of the central canal are round, oval, or triangular. Light and dark ependymal cells, depending on the density of the cytoplasm, were found. The light ependymal cells are fewer than the dark cells. The ependyma cytoplasm contained numerous mitochondria, filamentous structures, one or more well-developed Golgi-complexes, vesicles of the smooth endoplasmic reticulum, ribosomes, lysosomes, multivesicular bodies, profiles of the rough endoplasmic reticulum, large osmophilic bodies, and microtubules. The nuclei of the ependyma cells usually have a simple, regular round or oval shape. They occupy a relatively large portion of the cell volume and lie in the central or mediobasal position. Some of the nuclei show deep invaginations into the karyoplasm. Most of the mitochondria occupy mainly the supranuclear portion of the apical cytoplasm. There are of the crista-typ. Ribosomes occur free in the cytoplasm, but some attached to the profiles of the rough endoplasmic reticulum or being arranged as polysomes. The filamentous structures are generally prominent cytoplasmic components and are distributed at the apical, lateral, or basal region of the ependymocytes. They are grouped into bundles and arranged in parallel arrays. Some of these bundles reach the plasmamembrane at the free lumina of the central canal, others take contact to the filamentous structures of the zonulae adherentes of the junctional complex below the free surface. The granular endoplasmic reticulum shows specializations. There profiles surrounding granular substances and widely distributed granulations in connection with the nuclear envelope. The functional significance of the deposition of these granulations is still unknown. The luminal surface of the ependymocytes bears many microvilli and cilia. The cilia are regularly arranged in cranio-caudal direction. Each cilium has the typical (9 + 2)-subfibres. The intercellular space at the surface of the ependymal layer shows a single zonula adherens or zonulae adherentes in the row. Tight junctions and gap junctions were not found in the material examined. Cell processes of liquor contacting neurons between adjacent ependyma cells, protruding into the lumen of the central canal, could be observed. The termination of these neurons contains accumulations of mitochondria in the central part, large amounts of vesicles, and small dense bodies. They have short microvilli and some stereocilia at the free surface.(ABSTRACT TRUNCATED AT 400 WORDS)     

Low and high voltage electron microscopy of mitosis and cytokinesis in maize roots

The structure and distribution of cytoplasmic membranes during mitosis and cytokinesis in maize root tip meristematic cells was investigated by low and high voltage electron microscopy. The electron opacity of the nuclear envelope and endoplasmic reticulum (ER) was enhanced by staining the tissue in a mixture of zinc iodide and osmium tetroxide. Thin sections show the nuclear envelope to disassemble at prophase and become indistinguishable from the surrounding ER and polar aggregations of ER. In thick sections under the high voltage electron microscope the spindle is seen to be surrounded by a mass of tubular (TER) and cisternal (CER) endoplasmic reticulum derived from both the nuclear envelope and ER, which persists through metaphase and anaphase. At anaphase strands of TER traverse the spindle between the arms of the chromosomes. The octagonal nuclear pore complexes disappear by metaphase, but irregular-shaped pores persist in the membranes during mitosis. It is suggested that these form a template for pore-complex reformation during telophase. Phragmoplast formation is preceded by an aggregation of TER across the spindle at anaphase. Evidence is presented to suggest that the formation of the desmotubule of a plasmodesma is by the squeezing of a strand of endoplasmic reticulum between the vesicles of the cell plate.Abbreviations CER cisternal endoplasmic reticulum - ER endoplasmic reticulum - HVEM high voltage electron microscope - TER tubular endoplasmic reticulum - ZIO zinc iodide/osmium tetroxide     

Depletion of calcium from the lumen of endoplasmic reticulum reversibly inhibits passive diffusion and signal-mediated transport into the nucleus

Nuclear pore complexes provide channels for molecular transport across the nuclear envelope. Translocation of most proteins and RNAs through the pore complex is mediated by signal- and ATP-dependent mechanisms, while transport of small molecules is accomplished by passive diffusion. We report here that depletion of calcium from the lumen of the endoplasmic reticulum and nuclear envelope with ionophores or the calcium pump inhibitor thapsigargin rapidly and potently inhibits signal mediated transport of proteins into the nucleus. Lumenal calcium depletion also inhibits passive diffusion through the pore complex. Signal-mediated protein import and passive diffusion are rapidly restored when the drugs depleting lumenal calcium are removed and cells are incubated at 37 degrees C in calcium-containing medium. These results indicate that loss of calcium from the lumen of the endoplasmic reticulum and nuclear envelope reversibly affects properties of pore complex components located on the nuclear/cytoplasmic membrane surfaces, and they provide direct functional evidence for conformational flexibility of the pore complex. These methods will be useful for achieving reversible inhibition of nucleocytoplasmic trafficking for in vivo functional studies, and for studying the structure of the passive diffusion channel(s) of the pore complex.     

Ultrastructural localization of acetylcholinesterase activity in primary cultures of rat substantia nigra

Summary Ultrastructural localization of acetylcholinesterase activity was studied in primary cultures of the substantia nigra microdissected from newborn rat brains. Light microscopic observations were also made on the characteristics of dopamine neurones and acetylcholinesterase containing cells in these cultures. Ultrastructurally acetylcholinesterase activity was localized in the nuclear envelope and rough endoplasmic reticulum of neurones, which had deeply infolded, round or oval nucleus, a prominent Golgi apparatus and varying amounts of rough endoplasmic reticulum. In the neuropil acetylcholinesterase activity was seen within microtubules of neuronal processes and in the rough endoplasmic reticulum of dendrites. The enzyme activity was also demonstrated within the nuclear envelope and rough endoplasmic reticulum of probably capillary endothelial cells. Dopaminergic neurones were identified on the basis of the green catecholamine fluorescence they exhibited. Small dopaminergic neurones could be observed and there was indirect evidence that these cells did not stain for acetylcholinesterase.     

The Endoplasmic Reticulum and the Golgi Structures in Maize Root Cells

Maize root tips were fixed in potassium permanganate, embedded in epoxy resin, sectioned to show silver interference color, and studied with the electron microscope. All the cells were seen to contain an endoplasmic reticulum and apparently independent Golgi structures. The endoplasmic reticulum is demonstrated as a membrane-bounded, vesicular structure comparable in many aspects to that of several types of animal cells. With the treatment used here the membranes appear smooth surfaced. The endoplasmic reticulum is continuous with the nuclear envelope and, by contact at least, with structures passing through the cell wall. The nuclear envelope is characterized by discontinuities, as previously reported for animal cells. The reticula of adjacent cells seem to be in contact at or through the plasmodesmata. Because of these contacts the endoplasmic reticulum of a given cell appears to be part of an intercellular system. The Golgi structures appear as stacks of platelet-vesicles which apparently may, under certain conditions, produce small vesicles around their edges. Their form changes markedly with development of the cell.     

The endoplasmic reticulum and the Golgi structures in maize root cells

Maize root tips were fixed in potassium permanganate, embedded in epoxy resin, sectioned to show silver interference color, and studied with the electron microscope. All the cells were seen to contain an endoplasmic reticulum and apparently independent Golgi structures. The endoplasmic reticulum is demonstrated as a membrane-bounded, vesicular structure comparable in many aspects to that of several types of animal cells. With the treatment used here the membranes appear smooth surfaced. The endoplasmic reticulum is continuous with the nuclear envelope and, by contact at least, with structures passing through the cell wall. The nuclear envelope is characterized by discontinuities, as previously reported for animal cells. The reticula of adjacent cells seem to be in contact at or through the plasmodesmata. Because of these contacts the endoplasmic reticulum of a given cell appears to be part of an intercellular system. The Golgi structures appear as stacks of platelet-vesicles which apparently may, under certain conditions, produce small vesicles around their edges. Their form changes markedly with development of the cell.