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.     

Characterization of the structures involved in localization of the SUN proteins to the nuclear envelope and the centrosome

The nuclear envelope forms a selective barrier that separates the cytoplasm from the nucleus. During mitosis the nuclear envelope breaks down so that the microtubule network can form contacts with the kinetochore and guide chromosome segregation. Previous studies have suggested a model in which the centrosome and the microtubule network may play a role in nuclear envelope breakdown through as yet unidentified interactions with proteins localized to the nuclear envelope. In the current study we characterized a nuclear envelope protein SUN2 and identified a substructure involved in its localization to the nuclear envelope. We found that a structurally related protein, SUN1, may be localized to the nuclear envelope through a different mechanism. Furthermore, the SUN2 protein can form different assemblies, including homodimers and heterodimers with SUN1. Finally, we provide evidence indicating that SUN1 and SUN2 may form a physical interaction between the nuclear envelope and the centrosome.     

Cytoplasmic dynein as a facilitator of nuclear envelope breakdown.

During prophase in higher cells, centrosomes localize to deep invaginations in the nuclear envelope in a microtubule-dependent process. Loss of nuclear membranes in prometaphase commences in regions of the nuclear envelope that lie outside of these invaginations. Dynein and dynactin complex components concentrate on the nuclear envelope prior to any changes in nuclear envelope organization. These observations suggest a model in which dynein facilitates nuclear envelope breakdown by pulling nuclear membranes and associated proteins poleward along astral microtubules leading to nuclear membrane detachment. Support for this model is provided by the finding that interference with dynein function drastically alters nuclear membrane dynamics in prophase and prometaphase.     

Formation of the postmitotic nuclear envelope from extended ER cisternae precedes nuclear pore assembly

During mitosis, the nuclear envelope merges with the endoplasmic reticulum (ER), and nuclear pore complexes are disassembled. In a current model for reassembly after mitosis, the nuclear envelope forms by a reshaping of ER tubules. For the assembly of pores, two major models have been proposed. In the insertion model, nuclear pore complexes are embedded in the nuclear envelope after their formation. In the prepore model, nucleoporins assemble on the chromatin as an intermediate nuclear pore complex before nuclear envelope formation. Using live-cell imaging and electron microscope tomography, we find that the mitotic assembly of the nuclear envelope primarily originates from ER cisternae. Moreover, the nuclear pore complexes assemble only on the already formed nuclear envelope. Indeed, all the chromatin-associated Nup107-160 complexes are in single units instead of assembled prepores. We therefore propose that the postmitotic nuclear envelope assembles directly from ER cisternae followed by membrane-dependent insertion of nuclear pore complexes.     

Kinetochores on chromosomes enclosed within the nuclear envelope

The kinetochore plate which develops after nuclear envelope breakdown in normal cells can be seen to be formed on condensed chromosomes still enclosed in the nuclear envelope in fused multinucleate cells where some nuclei show delayed envelope breakdown caused by nuclear interaction. This suggests that neither nuclear envelope breakdown nor assembly of microtubules is directly related to the formation of the kinetochore plate. Furthermore, it can be clearly observed in these cells that the kinetochores do not have any special association with the nuclear envelope.     

Interaction of the activated cytoplasmic glucocorticoid hormone receptor complex with the nuclear envelope

Highly purified activated cytoplasmic glucocorticoid hormone receptor binds with high affinity to sites in the nuclear envelope. Nuclear envelope fragments can be isolated from purified chromatin. They bind activated cytoplasmic glucocorticoid receptor with the same equilibrium constant as nuclear envelopes. The presence of envelope components in chromatin is confirmed by the virtual identity of the gel electrophoretic glycoprotein pattern of nuclear envelope, chromatin nonhistones, and nuclear envelope fragments from chromatin.     

A freeze-fracture study of the nuclear envelope during spermiogenesis inMarsilea. Formation of a pore-free zone associated with the microtubule ribbon

Summary The nuclear envelope was freeze-fractured at different stages during spermiogenesis inMarsilea. Nuclear pores were seen at all stages of development, but were excluded from a specialized region of the nuclear envelope that is in contact with the microtubule ribbon. The microtubule ribbon is associated with one edge of the nuclear envelope throughout nuclear shaping and chromatin condensation and the pore-free zone may be a region of the nuclear envelope that is important in these processes.     

Identification of tyrosine-phosphorylated proteins associated with the nuclear envelope.

The nuclear envelope separates the nucleoplasm from the rest of the cell. Throughout the cell cycle, its structural integrity is controlled by reversible protein phosphorylation. Whereas its phosphorylation-dependent disassembly during mitosis is well characterized, little is known about phosphorylation events at this structure during interphase. The few characterized examples cover protein phosphorylation at serine and threonine residues, but not tyrosine phosphorylation at the nuclear envelope. Here, we demonstrate that tyrosine phosphorylation and dephosphorylation occur at the nuclear envelope of intact Neuro2a mouse neuroblastoma cells. Tyrosine kinase and phosphatase activities remain associated with purified nuclear envelopes. A similar pattern of tyrosine-phosphorylated nuclear envelope proteins suggests that the same tyrosine kinases act at the nuclear envelope of intact cells and at the purified nuclear envelope. We have also identified eight tyrosine-phosphorylated nuclear envelope proteins by 2D BAC/SDS/PAGE, immunoblotting with phosphotyrosine-specific antibodies, tryptic in-gel digestion, and MS analysis of tryptic peptides. These proteins are the lamina proteins lamin A, lamin B1, and lamin B2, the inner nuclear membrane protein LAP2beta, the heat shock protein hsc70, and the DNA/RNA-binding proteins PSF, hypothetical 16-kDa protein, and NonO, which copurify with the nuclear envelope.     

Chromosome ends and the nuclear envelope at premeiotic interphase in the male grasshopper Brachystola magna by 3-D,E.M. reconstruction

During premeiotic interphase in the male grasshopper Brachystola magna the nucleus is divided into two nuclear envelope bound compartments, one containing the X chromosome and one the autosomes. — The autosomal compartment is characterized by an invaginated nuclear envelope with nuclear pores distributed throughout the envelope. In a polarized region of the cell the pericentric heterochromatic chromocenters are associated with the inner membrane of the envelope invaginations. In this species the chromosomes are telocentric (acrocentric?) and the pericentric heterochromatin marks the proximal chromosome ends. It is concluded that the chromosome ends are attached to the nuclear envelope at premeiotic interphase. — Comparisons are made between the present observations on chromosome arrangements and the nuclear envelope at premeiotic interphase to earlier observations at early meiotic prophase in the same species (Church, 1976). It is concluded that a rearrangement of both the proximal chromosome ends and the nuclear envelope occurs as cells enter meiotic prophase.     

CRUCIFORM NUCLEAR DIVISION IN SOROSPHAERA VERONICAE

Sorosphaera veronicae Schroet. is an endobiotic, holocarpic, obligately parasitic fungus presently classified in the Plasmodiophoromycetes. The ultrastructure of nuclear envelope formation in somatic nuclear division in cystosoral plasmodia was studied. The inner membrane of the nuclear envelope during prophase appears to invaginate and blebb off intranuclear membranous vesicles. The intranuclear membranous vesicles become associated with the surface of the separating chromatin in anaphase and eventually are involved in the formation of daughter nuclear envelopes within the original nuclear envelope. The sequence of nuclear envelope breakdown and reformation in S. veronicae is noteworthy because it emphasizes alternate methods of nuclear envelope formation other than the generally considered “typical” formation described in Allium cepa L.     

Effects of brefeldin A on the Golgi apparatus, the nuclear envelope, and the endoplasmic reticulum in a green alga,Scenedesmus acutus

Summary The effects of brefeldin A (BFA) on the structure of the Golgi apparatus, the nuclear envelope, and the endoplasmic reticulum (ER), and on the thiamine pyrophosphatase (TPPase) activity in these organelles were examined in a green alga,Scenedesmus acutus, to obtain evidence for the existence of a retrograde transport from the Golgi apparatus to the ER via the nuclear envelope. InScenedesmus, Golgi bodies are situated close to the nuclear envelope throughout the cell cycle and receive the transition vesicles not directly from the ER, but from the nuclear envelope. BFA induced the disassembly of Golgi bodies and an increase in the ER cisternae at the trans-side of decomposed Golgi bodies in interphase cells and multinuclear cells before septum formation. The accumulated ER cisternae connected to the nuclear envelope at one part. TPPase activity was detected in all cisternae of Golgi bodies, but not in the nuclear envelope or the ER in nontreated cells. On the contrary, in BFA-treated cells, TPPase activity was detected in the nuclear envelope and the ER in addition to the decomposed Golgi bodies. When septum-forming cells were treated with BFA, the disassembly of Golgi bodies was less than that in interphase cells, and TPPase activity was detected in the Golgi cisternae but not in the nuclear envelope or the ER. These results suggest mat BFA blocks the anterograde transport from the nuclear envelope to the Golgi bodies but does not block the retrograde transport from the Golgi bodies to the nuclear envelope in interphase and multinuclear cells.Abbreviations BFA brefeldin A - ER endoplasmic reticulum - TPPase thiamine pyrophosphatase     

Dissociation of heterochromatin protein 1 from lamin B receptor induced by human polyomavirus agnoprotein: role in nuclear egress of viral particles

The nuclear envelope is one of the chief obstacles to the translocation of macromolecules that are larger than the diameter of nuclear pores. Heterochromatin protein 1 (HP1) bound to the lamin B receptor (LBR) is thought to contribute to reassembly of the nuclear envelope after cell division. Human polyomavirus agnoprotein (Agno) has been shown to bind to HP1alpha and to induce its dissociation from LBR, resulting in destabilization of the nuclear envelope. Fluorescence recovery after photobleaching showed that Agno increased the lateral mobility of LBR in the inner nuclear membrane. Biochemical and immunofluorescence analyses showed that Agno is targeted to the nuclear envelope and facilitates the nuclear egress of polyomavirus-like particles. These results indicate that dissociation of HP1alpha from LBR and consequent perturbation of the nuclear envelope induced by polyomavirus Agno promote the translocation of virions out of the nucleus.     

The subnuclear localization of tRNA ligase in yeast

Yeast tRNA ligase is an enzyme required for tRNA splicing. A study by indirect immune fluorescence shows that this enzyme is localized in the cell nucleus. At higher resolution, studies using indirect immune electron microscopy show this nuclear location to be primarily at the inner membrane of the nuclear envelope, most likely at the nuclear pore. There is a more diffuse, secondary location of ligase in a region of the nucleoplasm within 300 nm of the nuclear envelope. When the amount of ligase in the cell is increased, nuclear staining increases but staining of the nuclear envelope remains constant. This experiment indicates that there are a limited number of ligase sites at the nuclear envelope. Since the other tRNA splicing component, the endonuclease, has the characteristics of an integral membrane protein, we hypothesize that it constitutes the site for the interaction of ligase with the nuclear envelope.     

Characterization of the NAD+ glycohydrolase associated with the rat liver nuclear envelope.

The localization of NAD+ glycohydrolase [EC 3.2.2.5] (NADase) in purified rat liver nuclei has been examined. Subnuclear fractionation revealed that at least 70% of the NADase in nuclei was associated with the nuclear envelope fraction. The nuclear envelope fraction was practically free of microsomal contamination as judged by electron microscopic morphometry and assays of microsomal marker enzymes. Therefore, NADase was found to be an integral component of the nuclear envelope. The enzymological properties of the nuclear envelope NADase were compared with those of the microsomal enzyme. The nuclear envelope NADase was identical to the microsomal enzyme in its Km for NAD+ (60 muM), pH optimum (pH 6.5), ratio of transglycosidase activity to NADase activity (about 0.5), thermal stability and sensitivity to various inhibitors. Thus, NADase is a common enzymic component of both the nuclear envelope and the endoplasmic reticulum.     

Nucleocytoplasmic transport and nuclear envelope integrity in the fission yeast Schizosaccharomyces pombe

The nuclear envelope is essential for compartmentalizing the nucleus from the cytoplasm in all eukaryotic cells. There is a tremendous flux of both RNA and proteins across the nuclear envelope, which is intact throughout the entire cell cycle of yeasts but breaks down during mitosis of animal cells. Transport across the nuclear envelope requires the recognition of cargo molecules by receptors, docking at the nuclear pore, transit through the nuclear pore, and then dissociation of the cargo from the receptor. This process depends on the RanGTPase system, transport receptors, and the nuclear pore complex. We provide an overview of the nuclear transport process, with particular emphasis on the fission yeast Schizosaccharomyces pombe, including strategies for predicting and experimentally verifying the signals that determine the sub-cellular localization of a protein of interest. We also describe a variety of reagents and experimental strategies, including the use of mutants and chemical inhibitors, to study nuclear protein import, nuclear protein export, nucleocytoplasmic protein shuttling, and mRNA export in fission yeast. The RanGTPase and its regulators also play an essential transport independent role in nuclear envelope re-assembly after mitosis in animal cells and in the maintenance of nuclear envelope integrity at mitosis in S. pombe. Several experimental strategies and reagents for studying nuclear size, nuclear shape, the localization of nuclear pores, and the integrity of the nuclear envelope in living fission yeast cells are described.     

Identification and redistribution of lamins during nuclear differentiation in mouse spermatogenesis

Chromatin may be attached to the nuclear envelope through interaction of the nuclear membrane lamins A, B, and C. Such a hypothesis requires that these proteins are present in all cells with chromatin attachment to the nuclear envelope. We have investigated the distribution of the lamins during spermatogenesis in mouse, which exhibits extremes in nuclear envelope structural changes. By immunohistochemical techniques using human auto-antibodies and monoclonal antibodies against these molecules, we found that the lamins persist through all stages of spermatogenesis, though in highly variable amounts. They are also present during meiotic prophase (pachytene) when chromosomes are only locally attached to the nuclear envelope, analogous to the early prophase of somatic cells. Restructuring of the early spermatid nuclear envelope is accompanied by the appearance of a new lamin at the acrosomal fossa. In the epididymal spermatozoon the distribution of different lamins varies markedly over the nucleus suggesting special structural functions. The presence of lamins throughout spermatogenesis supports the concept that they are a general feature of the nuclear envelope structure, even where a lamina is not recognizable ultrastructurally.     

Centriolar plaque and spindle microtubules in the ascomyceteSordaria humana

Centriolar plaque and spindle microtubules in young asci of an ascomycete,Sordaria humana were studied by electron microscopy. Centriolar plaque is electron opaque and has an amorphous structure. Two dispositions of centriolar plaque were observed, one entirely contiguous to the nuclear envelope in a meiotic division and the other partially joined to the envelope in a mitotic division following meiosis. The spindle was formed inside the nuclear envelope and spindle microtubules terminated at the polar protrusion of the nuclear envelope. Some spindle microtubules seem to connect directly with the centriolar plaque passing through perforations of the nuclear envelope.     

Nuclear envelope disassembly and nuclear lamina depolymerization during germinal vesicle breakdown in starfish

During germinal vesicle breakdown (GVBD) in starfish, the nuclear envelope disassembles before the nuclear lamina completely depolymerizes, judging from correlative ultrastructural, immunolabeling, and light microscopic analyses. At 13 degrees C, prophase-arrested oocytes of Pisaster ochraceus begin GVBD and rapidly undergo nuclear envelope disassembly about 50 min after addition of the maturation-inducing hormone 1-methyladenine (1-MA). The nuclear lamina of these oocytes, however, remains present for 10-20 min following the vesiculation of the nuclear envelope. Completion of GVBD, as evidenced by a blending of the nuclear contents with the surrounding cytoplasm, occurs within about 15 min after the nuclear lamina has fully depolymerized. Immunofluorescence studies also indicate that a marked increase in the phosphorylations of nuclear proteins precedes the structural reorganizations of the nuclear envelope and nuclear lamina during GVBD.     

Membrane anchoring domain of herpes simplex virus glycoprotein gB is sufficient for nuclear envelope localization.

We have used the glycoprotein gB of herpes simplex virus type 1 (gB-1), which buds from the inner nuclear membrane, as a model protein to study localization of membrane proteins in the nuclear envelope. To determine whether specific domains of gB-1 glycoprotein are involved in localization in the nuclear envelope, we have used deletion mutants of gB-1 protein as well as chimeric proteins constructed by replacing the domains of the cell surface glycoprotein G of vesicular stomatitis virus with the corresponding domains of gB. Mutant and chimeric proteins expressed in COS cells were localized by immunoelectron microscopy. A chimeric protein (gB-G) containing the ectodomain of gB and the transmembrane and cytoplasmic domains of G did not localize in the nuclear envelope. When the ectodomain of G was fused to the transmembrane and cytoplasmic domains of gB, however, the resulting chimeric protein (G-gB) was localized in the nuclear envelope. Substitution of the transmembrane domain of G with the 69 hydrophobic amino acids containing the membrane anchoring domain of gB allowed the hybrid protein (G-tmgB) to be localized in the nuclear envelope, suggesting that residues 721 to 795 of gB can promote retention of proteins in the nuclear envelope. Deletion mutations in the hydrophobic region further showed that a transmembrane segment of 21 hydrophobic amino acids, residues 774 to 795 of gB, was sufficient for localization in the nuclear envelope. Since wild-type gB and the mutant and chimeric proteins that were localized in the nuclear envelope were also retained in the endoplasmic reticulum, the membrane spanning segment of gB could also influence retention in the endoplasmic reticulum.