Are accessory nuclei involved in the establishment of developmental gradients in hymenopteran oocytes?

Summary In the oocytes ofTenthredo olivacea, accessory nuclei (AN) are formed by budding from the nuclear envelope of the oocyte nucleus. Newly formed AN contain electron-dense material of nuclear origin and are surrounded by a double envelope devoid of pores. Such structures are subsequently transported to the peripheral ooplasm (periplasm), where they grow to reach a final diameter of 5 µm. In the envelopes of advanced AN nuclear pores arise. Through these pores nuage material is extruded into the surrounding periplasm. These findings are discussed with respect to a possible involvement of AN in the establishment of developmental gradients in hymenopteran oocytes.     

Unequal distribution of nuclear pores in oryzalin-induced mini-nuclei in protonema cells of the mossFunaria hygrometrica: influence of the nucleolus

Summary Mini-nuclei, formed in tip cells ofFunaria caulonemata after oryzalin treatment, have unequally distributed nuclear pores. The region of the nuclear envelope near the nucleolus, in a distance of up to 3 m, is devoid of pores. In other areas pores occur with a high frequency.     

Nuclear Import and Dimerization of Tomato ASR1, a Water Stress-Inducible Protein Exclusive to Plants

The ASR (for ABA/water stress/ripening) protein family, first described in tomato as nuclear and involved in adaptation to dry climates, is widespread in the plant kingdom, including crops of high agronomic relevance. We show both nuclear and cytosolic localization for ASR1 (the most studied member of the family) in histological plant samples by immunodetection, typically found in small proteins readily diffusing through nuclear pores. Indeed, a nuclear localization was expected based on sorting prediction software, which also highlight a monopartite nuclear localization signal (NLS) in the primary sequence. However, here we prove that such an "NLS" of ASR1 from tomato is dispensable and non-functional, being the transport of the protein to the nucleus due to simple diffusion across nuclear pores. We attribute such a targeting deficiency to the misplacing in that cryptic NLS of two conserved contiguous lysine residues. Based on previous in vitro experiments regarding quaternary structure, we also carried out live cell imaging assays through confocal microscopy to explore dimer formation in planta. We found homodimers in both the cytosol and the nucleus and demonstrated that assembly of both subunits together can occur in the cytosol, giving rise to translocation of preformed dimers. The presence of dimers was further corroborated by means of in vivo crosslinking of nuclei followed by SDS-PAGE.     

Nuclear pore clusters and perinuclear material in midgut epithelial cells of Dytiscus marginalis (Insecta, Coleoptera)

In ultrathin sections and freeze-fractures in hexagonal nuclear pore arrangement is described in midgut epithelial cells of Dytiscus marginalis. The majority of the pores is concentrated in several cup-like indentations of the nuclear envelope and the pore center-to-center distance is about 130 to 140 nm. In addition, in the regions of the nuclear pore clusters, patches of cytoplasm containing paracrystalline structures of closely packed hexagons of 18 to 20 nm width are found sandwiched between the nuclear envelope and a "secondary" envelope. In such areas, structures are commonly observed that suggest transfer of material from the nucleus to the cytoplasm, sometimes in a dumb-bell-shaped state and more often as slender filaments which migrate across the nuclear pores.     

Chromatin and nuclear envelope of freeze-fractured, neuronal interphase nuclei, resolved by scanning electron microscopy

High-resolution scanning electron microscopy (SEM) has previously been used to study intracellular detail, including chromatin. It has, however, been commonly carried out either on cellular subfractions or following extraction methods to visualize detail. In the work presented here, intracellular detail of neurons of the dorsal root was visualized in situ by viewing freeze-fracture faces obtained after hypotonic expansion. This procedure permits the detailed resolution, by SEM, of juxtanuclear and intranuclear detail to a degree impossible without hypotonic dispersal. In agreement with work previously reported, nuclear chromatin of these interphase cells presents largely as 30-nm fibers, with a next higher hierarchical structure imparted by swelling in magnesium chloride. Detailed analyses showed that particles as small as 10-nm nucleosomes comprising the 30-nm chromatin fiber could be resolved, with "end-on" views of such fibers showing 5 nucleosomes per helical turn of the fiber. Chromatin fibers positioned subjacent to nuclear pores, or associated with "nuclear spaces" communicating with nuclear pores, were frequently found to be resolved as clusters, in an apparently more decondensed conformation, rather than tightly coiled into the 30-nm fiber. In addition, details of the nuclear envelope, including nuclear pores and perinuclear filaments as well as membranes of the endoplasmic reticulum, decorated with ribosomes, were clearly resolved.     

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.     

Changes in volume,surface area,and frequency of nuclear pores on the vegetative nucleus of tobacco pollen in fresh,hydrated, and activated conditions

The vegetative nucleus (VN) of Nicotiana tabacum L. has been qualitatively and quantitatively studied in fresh, hydrated, and activated pollen. Techniques included the use of optical sectioning by confocal scanning laser microscopy to obtain volume and surface area measurements, and stereoscopic pairs; and freeze-etch electron microscopy to estimate the frequency of nuclear pores per m² in the vegetative nucleus. Several morphological changes were observed to occur in pollen grain nuclei during the early processes of tube growth. In freshly dehisced pollen grain, the vegetative and generative nuclei were side by side, but following hydration and activation of the grain, the elongated generative nucleus became partially surrounded by the vegetative nucleus. It was found that during hydration, the surface area of the vegetative nucleus increased and there was a decrease in the frequency of nuclear pores. The calculated total number of pores remained similar. After activation and pollen-tube growth, the vegetative nucleus retained the same surface area as in the hydrated state but the frequency of nuclear pores decreased; therefore, the calculated total number of pores was significantly lowered. When considered alongside complementary biochemical data, these morphological results indicate that RNA production in the vegetative nucleus decreases following germination.Abbreviations VN vegetative nucleus (nuclei) - GN generativenucleus - GC generative cell - CSLM confocal scanning laser microscope We acknowledge research support by the Biotechnology Action Programm of the Commission of European Communities, and CNR for the fellowship awarded to Dr. Wagner. We would also like to thank Mrs. C. Faleri for the expert technical help.     

Nuclear Envelope: Nanoarray Responsive to Aldosterone

Signalling between cytosol and nucleus is mediated by nuclear pores. These supramolecular complexes represent intelligent nanomachines regulated by a wide spectrum of factors. Among them, steroid hormones specifically interact with the pores and thus modify ion conductivity and macromolecule permeability of the nuclear envelope. In response to aldosterone the pores undergo dramatic changes in conformation, changes that depend on the nature of the transported cargo. Such changes can be imaged at the nanometer scale by using atomic force microscopy. Furthermore, steroid-induced macromolecule transport across the nuclear envelope causes osmotic water movements and nuclear swelling. Drugs that interact with intracellular steroid receptors (spironolactone) or with plasma membrane sodium channels (amiloride) inhibit swelling. Steroid hormone action is blocked when nuclear volume changes are prevented. This is shown in frog oocytes and human endothelial cells. In conclusion, nuclear pores serve as steroid-sensitive gates that determine nuclear activity.     

Phylogeny and molecular evolution of the DMC1 gene within the StH genome species in Triticeae (Poaceae)

To estimate the phylogeny and molecular evolution of a single-copy nuclear disrupted meiotic cDNA (DMC1) gene within the StH genome species, two DMC1 homoeologous sequences were isolated from nearly all the sampled StH genome species and were analyzed with those from seven diploid taxa representing the St and H genomes in Triticeae. Sequence diversity patterns and genealogical analysis suggested that (1) there is a close relationship among North American StH genome species; (2) the DMC1 gene sequences of the StH genome species from North America and Eurasia are evolutionarily distinct; (3) the StH genome polyploids have higher levels of sequence diversity in the St genome homoeolog than the H genome homoeolog; (4) the DMC1 sequence may evolve faster in the polyploid species than in the diploids; (5) high dN and dN/dS values in the St genome within polyploid species could be caused by low selective constraints or AT-biased mutation pressure. Our result provides some insight on evolutionary dynamics of duplicate DMC1 gene, the polyploidization events and phylogeny of the StH genome species.     

Heterogeneity of Dyggve-Melchior-Clausen dwarfism

Summary Sibs with apparent Dyggve-Melchior-Clausen (DMC) dwarfism and normal intelligence are described. Three other familial and 3 sporadic cases with DMC dwarfism and normal intelligence are known. Twelve familial and 9 sporadic cases are known with the usual combination of DMC dwarfism and severe mental retardation. Since the two conditions appear to breed true they seem to be genetically different. We propose to name the former Smith-McCort dwarfism to clearly distinguish it from the DMC syndrome in which mental retardation is a constituent part. Both conditions are inherited as autosomal recessive traits. Spinal cord compression due to atlantoaxial instability is a serious and preventable complication of both disorders.     

Sufficient Amounts of Functional HOP2/MND1 Complex Promote Interhomolog DNA Repair but Are Dispensable for Intersister DNA Repair during Meiosis in Arabidopsis

During meiosis, homologous recombination (HR) is essential to repair programmed DNA double-strand breaks (DSBs), and a dedicated protein machinery ensures that the homologous chromosome is favored over the nearby sister chromatid as a repair template. The HOMOLOGOUS-PAIRING PROTEIN2/MEIOTIC NUCLEAR DIVISION PROTEIN1 (HOP2/MND1) protein complex has been identified as a crucial factor of meiotic HR in Arabidopsis thaliana, since loss of either MND1 or HOP2 results in failure of DNA repair. We isolated two mutant alleles of HOP2 (hop2-2 and hop2-3) that retained the capacity to repair meiotic DSBs via the sister chromatid but failed to use the homologous chromosome. We show that in these alleles, the recombinases RADIATION SENSITIVE51 (RAD51) and DISRUPTED MEIOTIC cDNA1 (DMC1) are loaded, but only the intersister DNA repair pathway is activated. The hop2-2 phenotype is correlated with a decrease in HOP2/MND1 complex abundance. In hop2-3, a truncated HOP2 protein is produced that retains its ability to bind to DMC1 and DNA but forms less stable complexes with MND1 and fails to efficiently stimulate DMC1-driven D-loop formation. Genetic analyses demonstrated that in the absence of DMC1, HOP2/MND1 is dispensable for RAD51-mediated intersister DNA repair, while in the presence of DMC1, a minimal amount of functional HOP2/MND1 is essential to drive intersister DNA repair.     

Freeze-fracture studies of annulate lamellae in zebrafish oocytes

Summary The zebrafish oocyte contains prominent stacks of annulate lamellae (AL) located primarily in a subcortical position of the ooplasm. Many lamellae comprising a stack eventually exhibit continuity with the rough-surfaced endoplasmic reticulum which is present in abundance in larger oocytes. Pore structure of both AL and nuclear envelope (NE) was studied and compared by use of freeze-fracture electron microscopy. In freeze-fracture replicas, the NE and AL pores were easily distinguished, and a variety of fracture planes with respect to the stacked AL were generated. The pore diameter of NE and AL is similar (100nm). The number of nuclear pores varied from an average of 40 pores/m² in early stage oocytes to nearly double this number in later stage oocytes. For AL, the center-to-center spacing (120–130 nm) and the number of pores per square micrometer (56–67) did not change markedly regardless of oocyte developmental stage examined. Hexagonal packing of AL pores is a common feature. The AL pores have an angular margin with octagonal symmetry suggested in some cases. The AL pore interior contains fibrillar and particulate components and, depending upon the fracture plane, may appear to be filled with a plug of material. Both P- and E-membrane fracture faces of AL have a relative scarcity of intramembranous particles. The non-porous membranes that extend from the AL, however, have a higher concentration of intramembranous particles.     

Phylogeny and molecular evolution of the DMC1 gene in the polyploid genus Leymus (Triticeae: Poaceae) and its diploid relatives

The level and pattern of nucleotide variation in duplicate genes provide important information on the evolutionary history of polyploids and divergent processes between homoeologous loci within lineages. Leymus, a group of allopolyploid species with the NsXm genomes, is a perennial genus with a diverse array of morphology, ecology, and distribution in Triticeae. To estimate the phylogeny and molecular evolution of a single-copy DMC1 gene in Leymus and its diploid relatives,DMC1 homoeologous sequences were isolated from the sampled Leymus species and were analyzed with those from 30 diploid taxa representing 18 basic genomes in Triticeae. Sequence diversity patterns and genealogical analysis suggested that: (i) different Leymus species might derive their Ns genome from different Psathyrostachys species; (ii) Pseudoroegneria has contributed to the nuclear genome of some Leymus species, which might result from recurrent hybridization or incomplete lineage sorting; (iii) the Xm genome origin of Leymus could differ among species; (iv) rapid radiation and multiple origin might account for the rich diversity, numbers of species, and wide ecological adaptation of Leymus species; and (v) the DMC1 sequence diversity of the Ns genome in Leymus species was lower than that in the Psathyrostachys diploids, while the level of DMC1 sequence diversity in Leymus was higher than that in diploid Pseudoroegneria. Our results provide new insight on the evolutionary dynamics of duplicate DMC1 genes, polyploid speciation, and the phylogeny of Leymus species.     

Are annulate lamellae in the Drosophila embryo the result of overproduction of nuclear pore components?

Annulate lamellae are cytoplasmic organelles composed of stacked sheets of membrane containing pores that are structurally indistinguishable from nuclear pores. The functions of annulate lamellae are not well understood. Although they may be found in virtually any eucaryotic cell, they occur most commonly in transformed and embryonic tissues. In Drosophila, annulate lamellae are found in the syncytial blastoderm embryo as it is cleaved to form the cellular blastoderm. The cytological events of the cellularization process are well documented, and may be used as temporal landmarks when studying changes in annulate lamellae. By using morphometric techniques to analyze electron micrographs of embryos, we are able to calculate the number of pores per nucleus in nuclear envelopes and annulate lamellae during progressive stages of cellularization. We find that annulate lamellae pores remain at a low level while nuclear envelopes are expanding and acquiring pores in early interphase. Once nuclear envelopes are saturated with pores, however, the number of annulate lamellae pores increases more than 10-fold in 9 min. Over the next 30 min it gradually declines to the initial low level. On the basis of these results, we propose (a) that pore synthesis and assembly continues after nuclear envelopes have been saturated with pores; (b) that these supernumerary pores accumulate transiently in cytoplasmic annulate lamellae; and (c) that because these pores are not needed by the embryo they are subsequently degraded.     

TIME SEQUENCE OF NUCLEAR PORE FORMATION IN PHYTOHEMAGGLUTININ-STIMULATED LYMPHOCYTES AND IN HELA CELLS DURING THE CELL CYCLE

The time sequence of nuclear pore frequency changes was determined for phytohemagglutinin (PHA)-stimulated human lymphocytes and for HeLa S-3 cells during the cell cycle. The number of nuclear pores/nucleus was calculated from the experimentally determined values of nuclear pores/µ² and the nuclear surface. In the lymphocyte system the number of pores/nucleus approximately doubles during the 48 hr after PHA stimulation. The increase in pore frequency is biphasic and the first increase seems to be related to an increase in the rate of protein synthesis. The second increase in pores/nucleus appears to be correlated with the onset of DNA synthesis. In the HeLa cell system, we could also observe a biphasic change in pore formation. Nuclear pores are formed at the highest rate during the first hour after mitosis. A second increase in the rate of pore formation corresponds in time with an increase in the rate of nuclear acidic protein synthesis shortly before S phase. The total number of nuclear pores in HeLa cells doubles from ~2000 in G₁ to ~4000 at the end of the cell cycle. The doubling of the nuclear volume and the number of nuclear pores might be correlated to the doubling of DNA content. Another correspondence with the nuclear pore number in S phase is found in the number of simultaneously replicating replication sites. This number may be fortuitous but leads to the rather speculative possibility that the nuclear pore might be the site of initiation and/or replication of DNA as well as the site of nucleocytoplasmic exchange. That is, the nuclear pore complex may have multiple functions.     

RAD51 supports DMC1 by inhibiting the SMC5/6 complex during meiosis

Meiosis is a fundamental process for sexual reproduction in most eukaryotes and the evolutionarily conserved recombinases RADiation sensitive51 (RAD51) and Disrupted Meiotic cDNA1 (DMC1) are essential for meiosis and thus fertility. The mitotic function of RAD51 is clear, but the meiotic function of RAD51 remains largely unknown. Here we show that RAD51 functions as an interacting protein to restrain the Structural Maintenance of Chromosomes5/6 (SMC5/6) complex from inhibiting DMC1. We unexpectedly found that loss of the SMC5/6 partially suppresses the rad51 knockout mutant in terms of sterility, pollen inviability, and meiotic chromosome fragmentation in a DMC1-dependent manner in Arabidopsis thaliana. Biochemical and cytological studies revealed that the DMC1 localization in meiotic chromosomes is inhibited by the SMC5/6 complex, which is attenuated by RAD51 through physical interactions. This study not only identified the long-sought-after function of RAD51 in meiosis but also discovered the inhibition of SMC5/6 on DMC1 as a control mechanism during meiotic recombination.

RAD51 functions as an interacting protein to restrain the SMC5/6 complex from inhibiting DMC1 during meiosis.