Dynamic rearrangement of nucleoporins during fungal "open" mitosis

Mitosis in animals starts with the disassembly of the nuclear pore complexes and the breakdown of the nuclear envelope. In contrast to many fungi, the corn smut fungus Ustilago maydis also removes the nuclear envelope. Here, we report on the dynamic behavior of the nucleoporins Nup214, Pom152, Nup133, and Nup107 in this "open" fungal mitosis. In prophase, the nuclear pore complexes disassembled and Nup214 and Pom152 dispersed in the cytoplasm and in the endoplasmic reticulum, respectively. Nup107 and Nup133 initially spread throughout the cytoplasm, but in metaphase and early anaphase occurred on the chromosomes. In anaphase, the Nup107-subcomplex redistributed to the edge of the chromosome masses, where the new envelope was reconstituted. Subsequently, Nup214 and Pom152 are recruited to the nuclear pores and protein import starts. Recruitment of nucleoporins and protein import reached a steady state in G2 phase. Formation of the nuclear envelope and assembly of nuclear pores occurred in the absence of microtubules or F-actin, but not if both were disrupted. Thus, the basic principles of nuclear pore complex dynamics seem to be conserved in organisms displaying open mitosis.     

Altered nuclear pore diameters in G1-arrested cells of the yeast Saccharomyces cerevisiae.

Nuclear pores in cells of the yeast Saccharomyces cerevisiae were examined by using the freeze-fracture technique. Nuclear pore diameters in actively growing cells appear to be exclusively of the normal diameter (75 to 115 nm), whereas some pore diameters in abnormally small G1-arrested cells produced by nitrogen starvation are unusually wide (120 to 160 nm). There may be a correlation between nuclear pore size and nuclear envelope size, the larger pores tending to occur in the smaller envelopes. The finding suggests that nuclear pore diameter may not function in regulating the flow of informational molecules from nucleus to cytoplasm, but may be implicated in regulating the flow of substrates into the nucleus.     

ON THE FINE STRUCTURE AND COMPOSITION OF THE NUCLEAR ENVELOPE

Nuclei from nearly ripe eggs of Rana pipiens were isolated and cleaned in 0.1 M KCl. The whole nucleus was then digested to various degrees with ribonuclease or trypsin, followed by washing and fixation in either osmium tetroxide or potassium permanganate. The nuclear envelope was dissected off, placed on a grid, air dried, and compared with undigested controls in the electron microscope. Some envelopes were dehydrated, embedded in methacrylate, and sectioned. Annuli around "pores" are composed of a substance or substances, at least partially fibrillar, which is preserved by osmium but lost during permanganate fixation. Material within the "pores" is also preserved by osmium but partially lost after permanganate. No evidence of granules or tubules in the annuli was found in air dried mounts although a granular appearance could be seen in tangentially oriented thin sections. Thin sections of isolated envelopes give evidence of diffuse material within the "pores" as well as a more condensed diaphragm across their waists. In whole mounts of the envelope the total density within "pores" is relatively constant from "pore" to "pore." All material within "pores," including the condensed diaphragm, is removable by trypsin digestion. Wispy material from the "pore" structure projects into the nucleus and annular material extends into the cytoplasm. Both annular and diaphragm materials remain with the envelope when it is isolated and are thus considered a part of its structure, not merely evidences of material passing through. There is no evidence of ribonuclease-removable material in any part of the "pore" complex.     

Exploring the nuclear envelope of herpes simplex virus 1-infected cells by high-resolution microscopy

Herpesviruses are composed of capsid, tegument, and envelope. Capsids assemble in the nucleus and exit the nucleus by budding at the inner nuclear membrane, acquiring tegument and the envelope. This study focuses on the changes of the nuclear envelope during herpes simplex virus 1 (HSV-1) infection in HeLa and Vero cells by employing preparation techniques at ambient and low temperatures for high-resolution scanning and transmission electron microscopy and confocal laser scanning microscopy. Cryo-field emission scanning electron microscopy of freeze-fractured cells showed for the first time budding of capsids at the nuclear envelope at the third dimension with high activity at 10 h and low activity at 15 h of incubation. The mean number of pores was significantly lower, and the mean interpore distance and the mean interpore area were significantly larger than those for mock-infected cells 15 h after inoculation. Forty-five percent of nuclear pores in HSV-1-infected cells were dilated to more than 140 nm. Nuclear material containing capsids protrude through them into the cytoplasm. Examination of in situ preparations after dry fracturing revealed significant enlargements of the nuclear pore diameter and of the nuclear pore central channel in HSV-1-infected cells compared to mock-infected cells. The demonstration of nucleoporins by confocal microscopy also revealed fewer pores but focal enhancement of fluorescence signals in HSV-1-infected cells, whereas Western blots showed no loss of nucleoporins from cells. The data suggest that infection with HSV-1 alters the number, size, and architecture of nuclear pores without a loss of nucleoporins from altered nuclear pore complexes.     

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.     

Mechanisms and Signals for the Nuclear Import of Proteins

In eukaryotes, the nuclear membrane provides a physical barrier to the passive diffusion of macromolecules from and into the cytoplasm. Nucleocytoplasmic traffic occurs through highly specialized structures known as nuclear pores, and involves the participation of a special class of transport proteins. Active transport across the nuclear pores is an energy-dependent process that relies on the activity of Ran-GTPases both in the nuclear and cytoplasmic compartments. Nuclear import of proteins is an essential step in regulating gene expression and the replication cycle of several viruses. In this review, the key mechanisms, pathways, and models underlying the transport of proteins across nuclear pores are analysed.Key Words: Nuclear pore complex, nuclear localization signal, importin, nuclear transport.     

Ion channels in murine nuclei during early development and in fully differentiated adult cells

Summary The nuclear envelope functions as a selective barrier between nucleus and cytoplasm. During cycles of cell division the nuclear envelope repeatedly disassembles and re-associates. Presumably, each cycle re-establishes the functional and structural integrity of the nuclear envelope. After repeated rounds of cell division, as occurs during differentiation, the selectivity and configuration of the envelope may change. We compare the ionic conductance and the nuclear pore density in four types of murine nuclei: germinal vesicles in oocytes, pronuclei in zygotes, nuclei from two-cell blastomeres, and somatic cell nuclei from the liver. A large-conductance ion channel is present in all nuclear envelopes. Liver cell nuclei have a greater number of these channels than those from earlier developmental stages, and they also have a higher density of nuclear pores. In this article we hypothesize an association between the ion channels and the nuclear pores.     

Composition of the plant nuclear envelope: theme and variations

The nuclear envelope is the hallmark of all eukaryotic cells, separating the nucleoplasm from the cytoplasm. At the same time, the nuclear envelope allows for the controlled exchange of macromolecules between the two compartments through nuclear pores and presents a surface for anchoring and organizing cytoskeletal components and chromatin. Although our molecular understanding of the nuclear envelope in higher plants is only just beginning, fundamental differences from the animal nuclear envelope have already been found. This review provides an updated investigation of these differences with respect to nuclear pore complexes, targeting of Ran signalling to the nuclear envelope, inner nuclear envelope proteins, and the role and fate of the nuclear envelope during mitosis.     

Ultrastructural analysis of the replication cycle of pseudorabies virus in cell culture: a reassessment.

We reinvestigated major steps in the replicative cycle of pseudorabies virus (PrV) by electron microscopy of infected cultured cells. Virions attached to the cell surface were found in two distinct stages, with a distance of 12 to 14 nm or 6 to 8 nm between virion envelope and cell surface, respectively. After fusion of virion envelope and cell membrane, immunogold labeling using a monoclonal antibody against the envelope glycoprotein gE demonstrated a rapid drift of gE from the fusion site, indicating significant lateral movement of viral glycoproteins during or immediately after the fusion event. Naked nucleocapsids in the cytoplasm frequently appeared close to microtubules prior to transport to nuclear pores. At the nuclear pore, nucleocapsids invariably were oriented with one vertex pointing to the central granulum at a distance of about 40 nm and viral DNA appeared to be released via the vertex region into the nucleoplasm. Intranuclear maturation followed the typical herpesvirus nucleocapsid morphogenesis pathway. Regarding egress, our observations indicate that primary envelopment of nucleocapsids occurred at the inner leaflet of the nuclear membrane by budding into the perinuclear cisterna. This nuclear membrane-derived envelope exhibited a smooth surface which contrasts the envelope obtained by putative reenvelopment at tubular vesicles in the Golgi area which is characterized by distinct surface projections. Loss of the primary envelope and release of the nucleocapsid into the cytoplasm appeared to occur by fusion of envelope and outer leaflet of the nuclear membrane. Nucleocapsids were also found engulfed by both lamella of the nuclear membrane. This vesiculation process released nucleocapsids surrounded by two membranes into the cytoplasm. Our data also indicate that fusion between the two membranes then leads to release of naked nucleocapsids in the Golgi area. Egress of virions appeared to occur via transport vesicles containing one or more virus particles by fusion of vesicle and cell membrane. Our data thus support biochemical data and mutant virus studies of (i) two steps of attachment, (ii) the involvement of microtubules in the transport of nucleocapsids to the nuclear pore, and (iii) secondary envelopment in the trans-Golgi area in PrV infection.     

Reconstitution of biochemically altered nuclear pores: transport can be eliminated and restored

Biochemically altered nuclear pores specifically lacking the N-acetylglucosamine-bearing pore proteins were constructed in a nuclear assembly extract in order to assign function to these proteins. The depleted pores do not bind nuclear signal sequences or actively import nuclear proteins, but they are functional for diffusion. These defects can be fully repaired by assembly with readded Xenopus pore glycoproteins. Strikingly, isolated rat pore glycoproteins also restore transport. Electron microscopy reveals that depleted pores have largely normal morphology. Thus, the pore glycoproteins are not required for assembly of the nuclear envelope, the major structures of the pore, or a pore diffusional channel. Instead, they are essential for active protein import and, unexpectedly, for construction of the part of the pore necessary for signal sequence recognition.     

SOME OBSERVATIONS ON THE NUCLEAR ENVELOPE

In maize root meristem cells, fixed in KMnO₄, embedded in epoxy resin, ultrathin sectioned, and studied with an electron microscope, the nuclear envelope is demonstrated to be a double membrane structure. In the nuclear envelope there are: pores of the sort reported in many species of animals and plants; different types of openings associated with extensions of both nuclear membranes into the cytoplasm; and also, often, large discontinuities. The nuclear envelope is a component of the general vesicular reticulum. The reticula of neighboring cells including the nuclear envelopes make up, at certain stages at least, a "systemic" structure. The status of the nuclear envelope as a component of the general cellular reticulum is recognized to change during differentiation. The existence of several types of discontinuities in the nuclear envelope and the extent of nuclear-cytoplasmic surface relationships indicated suggests alteration in concepts of transport and exchanges between nucleus and cytoplasm.     

Structure of the nuclear pore complex in mammalian cells. Two annular components

The ultrastructure of the nuclear pore complex has been investigated in isolated nuclei of an in vitro cultured bovine liver cell line. In shadow-cast replicas of the surface of nuclei isolated in Tris buffer containing low K⁺ and Mg²⁺ concentrations (RSB) the rims of the pores appeared as annular projections with an outer diameter of 100 to 120 nm. When the nuclei were isolated in Tris buffer containing 0.1% Triton the projections were essentially lost, together with the outer membrane of the nuclear envelope. In electron micrographs of whole-mount preparations the Triton-Tris nuclei—but not the RSB nuclei—were surrounded by numerous circular structures, which obviously had been detached from the nuclear surface during the preparation. They consisted of eight granules of about 20 nm diameter which were connected in a circular fashion by fibrous material. The circular structures had an inside diameter close to 65 nm. In broken nuclei many of these circular structures contained a second, smaller circular component and a central granule. From these observations it is concluded that the annulus of the nuclear pore consists of two components and that the outer component is located in the perinuclear space in intimate association with the membrane limiting the pore. A modified model of the nuclear pore complex which accounts for this location is proposed.     

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.     

Purification of the vertebrate nuclear pore complex by biochemical criteria

The nuclear pore is a large and complex biological machine, mediating all signal-directed transport between the nucleus and the cytoplasm. The vertebrate pore has a mass of ∼120 million daltons or 30 times the size of a ribosome. The large size of the pore, coupled to its tight integration in the nuclear lamina, has hampered the isolation of pore complexes from vertebrate sources. We have now developed a strategy for the purification of nuclear pores from in vitro assembled annulate lamellae (AL), a cytoplasmic mimic of the nuclear envelope that lacks a lamina, nuclear matrix, and chromatin-associated proteins. We find that purified pore complexes from annulate lamellae contain every nuclear pore protein tested. In addition, immunoblotting reveals the presence of soluble transport receptors and factors known to play important roles in the transport of macromolecules through the pore. While transport factors such as Ran and NTF2 show only transient interaction with the pores, a number of soluble transport receptors, including importin β, show a tight association with the purified pores. In summary, we report that we have purified the vertebrate pore by biochemical criteria; silver staining reveals ∼40–50 distinct protein bands.     

RNA TRANSPORT FROM NUCLEUS TO CYTOPLASM IN CHIRONOMUS SALIVARY GLANDS

The fine structure and cytochemistry of the extremely large RNA puffs, or Balbiani rings, in salivary gland nuclei of midge, Chironomus thummi, larvae have been investigated. The Balbiani rings are composed of a diffuse mass of electron-opaque 400 to 500 A granules, short threads about 180 to 220 A in diameter and associated fine chromatin fibrils. These components appear to be organized into brushlike elements which form the ring. Electron microscope cytochemistry has shown that the granules and short threads contain RNA. After ribonuclease digestion, only 50 to 100 A chromatin fibrils were apparent in the Balbiani ring, and the granules were no longer demonstrable. Deoxyribonuclease digestion had no apparent effect on these structures. Observations indicate that the granules are formed from the short threads and released into the nucleoplasm in which they are evenly distributed. At the nuclear envelope, many granules have been observed partially or completely within the nuclear pores. These granules become elongated and are shown to penetrate the center of the pore in a rodlike form, about 200 A in diameter. The Balbiani ring granules are not normally visible within the cytoplasm adjacent to the nuclear envelope, but have been rarely found in this region. It is suggested that the granules represent the product of the Balbiani ring, possibly a messenger RNA bound to protein, and that they regularly pass into the cytoplasm through a narrow central channel in the pores of the nuclear envelope.     

Evidence for changes in nuclear permeability during different physiological states

It was found that the nuclei of well-fed amoebae accumulate colloidal gold from the cytoplasm at a significantly greater rate than the nuclei of cells starved for five or nine days. The results are most likely due to a decrease in the permeability of the nuclear envelope during starvation. Evidence was obtained indicating that the permeability decrease is caused by a change in the functional properties of the pores, rather than a change in either pore size or number. These findings are consistent with the view that the nuclear pores are involved in regulating cellular activity.     

Early localization of NPA58, a rat nuclear pore-associated protein, to the reforming nuclear envelope during mitosis

We have studied the mitotic reassembly of the nuclear envelope, using antibodies to nuclear marker proteins and NPA58 in F-111 rat fibroblast cells. In earlier studies we have proposed that NPA58, a 58 kDa rat nuclear protein, is involved in nuclear protein import. In this report, NPA58 is shown to be localized on the cytoplasmic face of the envelope in interphase cells, in close association with nuclear pores. In mitotic cells NPA58 is dispersed in the cytoplasm till anaphase. The targeting of NPA58 to the reforming nuclear envelope in early telophase coincides with the recruitment of a well-characterized class of nuclear pore proteins recognized by the antibody mAb 414, and occurs prior to the incorporation of lamin B1 into the envelope. Significant protein import activity is detectable only after localization of NPA58 in the newly-formed envelope. The early targeting of NPA58 is consistent with its proposed role in nuclear transport.