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PubMed2005

The myristoylation site of meiotic lamin C2 promotes local nuclear membrane growth and the formation of intranuclear membranes in somatic cultured cells.

Lamin C2 is a splice product of the mammalian lamin A gene and expressed in primary spermatocytes where it is distributed in the form of discontinuous plaques at the nuclear envelope. We have previously shown that the aminoterminal hexapetide GNAEGR of lamin C2 following the start methionine is essential for its association with the nuclear envelope and that the aminoterminal glycine of the hexapeptide is myristoylated. Here we have analyzed the ultrastructural changes induced in COS-7 and Xenopus A6 cells by overexpressing rat lamin C2 or a human lamin C mutant possessing the lamin C2-specific hexapeptide at its aminoterminus. Both lamins were targeted to the nuclear envelope of mammalian and amphibian cells and induced the formation of intranuclear membranes, whereas wild-type human lamin C and a lamin C2 mutant, that both lack this lipid moiety, did not. Our data indicate that the myristoyl group of lamin C2 has besides its demonstrated role in nuclear envelope association additional functions during spermatogenesis. Our present study complements previously published results where we have shown that the CxxM motif of lamins promotes nuclear membrane growth (Prüfert et al., 2004. J. Cell Sci. 117, 6105-6116).

Animals↗
PubMed2005

Pushing the envelope: structure, function, and dynamics of the nuclear periphery.

The nuclear envelope (NE) is a highly specialized membrane that delineates the eukaryotic cell nucleus. It is composed of the inner and outer nuclear membranes, nuclear pore complexes (NPCs) and, in metazoa, the lamina. The NE not only regulates the trafficking of macromolecules between nucleoplasm and cytosol but also provides anchoring sites for chromatin and the cytoskeleton. Through these interactions, the NE helps position the nucleus within the cell and chromosomes within the nucleus, thereby regulating the expression of certain genes. The NE is not static, rather it is continuously remodeled during cell division. The most dramatic example of NE reorganization occurs during mitosis in metazoa when the NE undergoes a complete cycle of disassembly and reformation. Despite the importance of the NE for eukaryotic cell life, relatively little is known about its biogenesis or many of its functions. We thus are far from understanding the molecular etiology of a diverse group of NE-associated diseases.

Animals↗
PubMed2004

Ultrastructural localization of thyroid peroxidase, hydrogen peroxide-generating sites, and monoamine oxidase in benign and malignant thyroid diseases.

Despite thyroid tissue heterogeneity, biochemical and morphological features have been associated with certain thyroid diseases. We analyzed the ultracytochemical localization of thyroperoxidase (TPO), TPO-associated hydrogen peroxide-generating sites (H(2)O(2) sites), and monoamine oxidase (MAO) in terms of morphology and biochemical TPO activity in abnormal thyroids. We examined 11 cases of nontoxic multinodular goiter, 5 cases of Hashimoto's thyroiditis, 1 case of oncocytic (Hürthle or oxyphilic cell) adenoma, 5 cases of Graves' disease, 4 cases of papillary carcinoma, and 4 cases of perinodular normal tissue. In the perinodular tissue, TPO was detected mainly in the nuclear envelope, rough endoplasmic reticulum (RER), and subapical vesicles, but not in the apical surface. In multinodular goiter, heterogeneous TPO reactivity ranging from almost null to strongly positive was detected in similar locations as in the perinodular tissue, and was absent in the microvilli. Follicular cells from Hashimoto's thyroiditis displayed TPO in the nuclear envelope and the scarce RER. Remarkably, oncocytic cells from both Hashimoto's thyroiditis and oncocytic adenoma, typically packed with mitochondria, displayed evident TPO reaction exclusively in mitochondrial cristae. In Graves' disease, the nuclear envelope, enlarged RER, and apical vesicles were strongly TPO positive, and microvilli also exhibited TPO activity. Papillary carcinoma cells were negative for TPO. The localization and characteristics of TPO activity in the H(2)O(2) sites were similar to that of TPO in all tissues. MAO was positive in mitochondria of perinodular tissues, multinodular goiter, and oncocytes and negative in Hashimoto's thyroiditis and Graves' disease. Interestingly, MAO was intensely positive in the nuclear envelope of papillary carcinoma but unreactive in mitochondria. Biochemical TPO activity was increased in multinodular goiter and Graves' disease. In conclusion, several changes in ultracytochemical characteristics of TPO, H(2)O(2) sites, and MAO were associated with thyroid disease. Nonmalignant oncocytic cells exhibited an unusual mitochondrial location of TPO and H(2)O(2) sites. The distribution of MAO in nuclear envelope of papillary carcinoma cells could be a further feature of malignancy.

Humans↗
PubMed1980

Evidence from rat liver nuclear preparations that latency of microsomal UDP-glucuronosyltransferase is associated with vesiculation.

1. Nuclear, nuclear-envelope and microsomal preparations were prepared from rat liver, and their purity and morphology monitored by electron microscopy. 2. UDP-glucuronosyltransferase activity in microsomal preparations, but not in standard nuclear or nuclear-envelope preparations, displays latency from the criterion of being enhanced ('activated') by a range of detergents or the endogenous activator UDP-N-acetyl-glucosamine. 3. Nuclear preparations resemble activated rather than native microsomal preparations in failing to transfer glucuronic acid from 4-nitrophenyl glucuronide to 2-aminophenol. 4. Electron microscopy indicates that membranes of nuclear preparations and of our standard nuclear-envelope preparations remain, as in vivo, in a cisternal arrangement, whereas those of microsomal preparations are vesiculated. 5. In nuclear-envelope preparations in which vesiculation has been encouraged, the transferase can be activated by detergents. 6. We suggest that latency of UDP-glucuronosyltransferase results from vesiculation of membranes during preparation and that the latency of the microsomal transferase is largely a preparative artefact.

Aminophenols↗
PubMed1984

Endogenous peroxidase activity in mononuclear phagocytes.

The diaminobenzidine (DAB) technique has been used to visualize the subcellular localization of peroxidatic enzymes in mononuclear phagocytes. The latter cells are part of the mononuclear phagocyte system (MPS), which includes the monocytes in the bone marrow and blood, their precursors in the bone marrow, and the resident macrophages in the tissues. The DAB cytochemistry has revealed distinct subcellular distribution patterns of peroxidase in the mononuclear phagocytes. Thus the technique facilitates the identification of the various phagocyte types: Promonocytes contain peroxidase reaction in the nuclear envelope, endoplasmic reticulum, Golgi apparatus, and cytoplasmic granules. Monocytes exhibit the reaction product only in cytoplasmic granules. Most resident macrophages show the activity only in the nuclear envelope and endoplasmic reticulum. Furthermore, new phagocyte types have been detected based on the peroxidase cytochemistry. Intermediate cells between monocytes and resident macrophages contain reaction product in the nuclear envelope, endoplasmic reticulum and cytoplasmic granules. The resident macrophages can be divided into two subtypes. Most of them exhibit the pattern noted above. Some, however, are totally devoid of peroxidase reaction. Most studies on peroxidase cytochemistry of monocytes and macrophages agree that the peroxidase patterns reflect differentiation or maturation stages of one cell line. Some authors, however, still interpret the patterns as invariable characteristics of separate cell lines. As to the function of the peroxidase in phagocytes, the cytochemical findings imply that two different peroxidatic enzymes exist in the latter cells: one peroxidase is synthesized in the endoplasmic reticulum of promonocytes and transported to granules via the Golgi apparatus. The synthesis ceases when the promonocyte matures to the monocyte. Upon phagocytosis the peroxidase is discharged into the phagosomes. Biochemical and functional studies have indicated that this peroxidase (myeloperoxidase) is part of a microbicidal system operating in host defence mechanisms. The other enzyme with peroxidatic activity is confined to the nuclear envelope and endoplasmic reticulum of resident macrophages in-situ and of monocytes at early stages in culture. As suggested by the subcellular distribution, the inhibition by peroxidase blockers, and the localization during phagocytosis studies, the latter peroxidase is functionally different from the myeloperoxidase.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed2006

Mechanical properties of the cell nucleus and the effect of emerin deficiency.

Nuclear structure and mechanics are gaining recognition as important factors that affect gene expression, development, and differentiation in normal function and disease, yet the physical mechanisms that govern nuclear mechanical stability remain unclear. Here we examined the physical properties of the cell nucleus by imaging fluorescently labeled components of the inner nucleus (chromatin and nucleoli) and the nuclear envelope (lamins and membranes) in nuclei deformed by micropipette aspiration (confocal imaged microdeformation). We investigated nuclei, both isolated and in intact, living cells, and found that nuclear volume significantly decreased by 60-70% during aspiration. While nuclear membranes exhibited blebbing and fluid characteristics during aspiration, the nuclear lamina exhibited behavior of a solid-elastic shell. Under large deformations of GFP-lamin A-labeled nuclei, we observed a decay of fluorescence intensity into the tip of the deformed tongue that we interpreted in terms of nonlinear, two-dimensional elasticity theory. Here we applied this method to study nuclear envelope stability in disease and found that mouse embryo fibroblasts lacking the inner nuclear membrane protein, emerin, had a significantly decreased ratio of the area expansion to shear moduli (K/mu) compared to wild-type cells (2.1 +/- 0.2 versus 5.1 +/- 1.3). These data suggest that altered nuclear envelope elasticity caused by loss of emerin could contribute to increased nuclear fragility in Emery-Dreifuss muscular dystrophy patients with mutations in the emerin gene. Based on our experimental results and theoretical considerations, we present a model describing how the nucleus is stabilized in the pipette. Such a model is essential for interpreting the results of any micropipette study of the nucleus and porous materials in general.

Animals↗
PubMed1976

The meotic prophase in Bombyx mori females analyzed by three dimensional reconstructions of synaptonemal complexes.

Serial sectioning followed by three dimensional reconstruction of lateral componenets of the synaptoemal complex have been used to follow chromosome pairing during theprophase of the achiasmatic meiotic division in the silkworm. Bombyx mori. During leptotene and early zygotene, the lateral components become attached to the nuclear envelope at a specific region, thus forming a chromosome bouquet. The attachment of lateral componenets to the nuclear envelope precedes the completion of the components between their attachment points. Synapsis and synaptonemal complex formation start during the period of lateral component organization in the individual nucleus. Telomeric movements on the nuclear envelope occur at two stages of the prophase: the chromosome pairing appears to be initiated by an association of unpaired ends of homologus chromosomes, the nature of this primary attraction and recognition being unknown. Secondly, the paired chromosomes become dispersed in the nucleus by shifting of attachment sites of completed synaptonemal complexes at the end of zygotene. This movement is possible related to a membranes flow occuring during this stage. Membrane material is synthesized at the region of synaptonemal complex attachment. Later, the excess membrane material is shifted to the opposite pole where it protudes into the lumen of the nuclei thus forming vacuoles. Two previously undescribed features of chromosomes paring were revealed. In late zygotene, chromosome pairing and synaptonmal complex formation were frequently observed to be delayed or even prevented over s short distance by interlocking two bivalents, both being attached to the nuclear envelope. Such interlocking of bivalents was not found in pachytene...

Animals↗
PubMed1997

Two proteins that cycle asynchronously between centrosomes and nuclear structures: Drosophila CP60 and CP190.

Both the nucleus and the centrosome are complex, dynamic structures whose architectures undergo cell cycle-specific rearrangements. CP190 and CP60 are two Drosophila proteins of unknown function that shuttle between centrosomes and nuclei in a cell cycle-dependent manner. These two proteins are associated in vitro, and localize to centrosomes in a microtubule independent manner. We injected fluorescently labeled, bacterially expressed CP190 and CP60 into living Drosophila embryos and followed their behavior during the rapid syncytial blastoderm divisions (nuclear cycles 10-13). Using quantitative 3-D wide-field fluorescence microscopy, we show that CP190 and CP60 cycle between nuclei and centrosomes asynchronously with the accumulation of CP190 leading that of CP60 both at centrosomes and in nuclei. During interphase, CP190 is found in nuclei. Immediately following nuclear envelope breakdown, CP190 localizes to centrosomes where it remains until telophase, thereafter accumulating in reforming nuclei. Unlike CP190, CP60 accumulates at centrosomes primarily during anaphase, where it remains into early interphase. During nuclear cycles 10 and 11, CP60 accumulates in nuclei simultaneous with nuclear envelope breakdown, suggesting that CP60 binds to an unknown nuclear structure that persists into mitosis. During nuclear cycles 12 and 13, CP60 accumulates gradually in nuclei during interphase, reaching peak levels just before nuclear envelope breakdown. Once in the nucleus, both CP190 and CP60 appear to form fibrous intranuclear networks that remain coherent even after nuclear envelope breakdown. The CP190 and CP60 networks do not co-localize extensively with each other or with DNA. This work provides direct evidence, in living cells, of a coherent protein network that may represent a nuclear skeleton.

Animals↗
PubMed2003

Nuclear pore protein gp210 is essential for viability in HeLa cells and Caenorhabditis elegans.

Gp210 is an evolutionarily conserved membrane protein of the nuclear pore complex (NPC). We studied the phenotypes produced by RNAi-induced downregulation of gp210 in both human (HeLa) cells and Caenorhabditis elegans embryos. HeLa cell viability requires Gp210 activity. The dying cells accumulated clustered NPCs and aberrant membrane structures at the nuclear envelope, suggesting that gp210 is required directly or indirectly for nuclear pore formation and dilation as well as the anchoring or structural integrity of mature NPCs. Essential roles for gp210 were confirmed in C. elegans, where RNAi-induced reduction of gp210 caused embryonic lethality. The nuclear envelopes of embryos with reduced gp210 also had aberrant nuclear membrane structures and clustered NPCs, confirming that gp210 plays critical roles at the nuclear membrane through mechanisms that are conserved from nematodes to humans.

Animals↗
PubMed2003

Nuclear phosphoinositide 3-kinase C2beta activation during G2/M phase of the cell cycle in HL-60 cells.

The activity of nuclear phosphoinositide 3-kinase C2beta (PI3K-C2beta) was investigated in HL-60 cells blocked by aphidicolin at G(1)/S boundary and allowed to progress synchronously through the cell cycle. The activity of immunoprecipitated PI3K-C2beta in the nuclei and nuclear envelopes showed peak activity at 8 h after release from the G(1)/S block, which correlates with G(2)/M phase of the cell cycle. In the nuclei and nuclear envelopes isolated from HL-60 cells at 8 h after release from G(1)/S block, a significant increase in the level of incorporation of radiolabeled phosphate into phosphatidylinositol 3-phosphate (PtdIns(3)P) was observed with no change in the level of radiolabeled PtdIns(4)P, PtdIns(4,5)P(2) and PtdIns(3,4,5)P(3). On Western blots, PI3K-C2beta revealed a single immunoreactive band of 180 kDa, whereas in the nuclei and nuclear envelopes isolated at 8 h after release, the gel shift of 18 kDa was observed. When nuclear envelopes were treated for 20 min with mu-calpain in vitro, the similar gel shift and increase in PI3K-C2beta activity was observed which was completely inhibited by pretreatment with calpain inhibitor calpeptin. The presence of PI3K inhibitor LY 294002 completely abolished the calpain-mediated increase in the activity of PI3K-C2beta but did not prevent the gel shift. When HL-60 cells were released from G(1)/S block in the presence of either calpeptin or LY 294002, the activation of nuclear PI3K-C2beta was completely inhibited. These results demonstrate the calpain-mediated activation of the nuclear PI3K-C2beta during G(2)/M phase of the cell cycle in HL-60 cells.

Aphidicolin↗
PubMed1985

Nucleocytoplasmic RNA transport.

A number of closely related post-transcriptional facets of RNA metabolism show nuclear compartmentation, including capping, methylation, splicing reactions, and packaging in ribonucleoprotein particles (RNP). These nuclear 'processing' events are followed by the translocation of the finished product across the nuclear envelope. Due to the inherent complexity of these interrelated events, in vitro systems have been designed to examine the processes separately, particularly so with regard to translocation. A few studies have utilized nuclear transplantation/microinjection techniques and specialized systems to show that RNA transport occurs as a regulated phenomenon. While isolated nuclei swell in aqueous media and dramatic loss of nuclear protein is associated with this swelling, loss of RNA is not substantial, and most studies on RNA translocation have employed isolated nuclei. The quantity of RNA transported from isolated nuclei is related to hydrolysis of high-energy phosphate bonds in nucleotide additives. The RNA is released predominantly in RNP: messenger-like RNA is released in RNP which have buoyant density and polypeptide composition similar to cytoplasmic messenger RNP, but which have distinctly different composition from those in heterogeneous nuclear RNP. Mature 18 and 28S ribosomal RNA is released in 40 and 60S RNP which represent mature ribosomal subunits. RNA transport proceeds with characteristics of an energy-requiring process, and proceeds independently of the presence or state of fluidity of nuclear membranes. The energy for transport appears to be utilized by a nucleoside triphosphatase (NTPase) which is distributed mainly within heterochromatin at the peripheral lamina. Photoaffinity labeling has identified the pertinent NTPase as a 46 kD polypeptide which is associated with nuclear envelope and matrix preparations. The NTPase does not appear to be modulated via direct phosphorylation or to reflect kinase-phosphatase activities. A large number of additives (including RNA and insulin) produce parallel effects upon RNA transport and nuclear envelope NTPase, strengthening the correlative relationship between these activities. Of particular interest has been the finding that carcinogens induce specific, long-lasting increases in nuclear envelope (and matrix) NTPase; this derangement may underlie the alterations in RNA transport associated with cancer and carcinogenesis.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1994

Indolic compounds affect tryptophan binding to rat hepatic nuclei.

This study evaluates the effects of indolic or indole-related compounds on binding of L-tryptophan (saturable, stereospecific and of high affinity) to rat hepatic nuclei or nuclear envelopes. Addition of any one of many indolic or indole-related compounds, and particularly of 3-methylindole, does not inhibit in vitro binding of [3H]tryptophan to hepatic nuclear envelopes. However, when 3-methylindole (10(-10) to 10(-4) mol/L) is added in combination with unlabeled L-tryptophan (10(-4) mol/L), it diminishes the inhibitory effect of unlabeled L-tryptophan alone. Scatchard analysis of the binding affinities of in vitro [3H]tryptophan binding to hepatic nuclear envelopes using L-tryptophan in the absence or presence of 3-methylindole reveals similar dissociation constants (KD) under the two conditions, but the binding concentrations (Bmax) were greater in the combined group compared with that in the L-tryptophan alone group. Addition of 3-methylindole to liver before homogenization decreases specific [3H]tryptophan binding to nuclei compared with controls (without addition). L-Tryptophan tube-fed to rats with or without 3-methylindole administration increases in vitro hepatic protein synthesis compared with that of saline tube-fed controls. 3-Methylindole itself does not affect protein synthesis. Our report describes the effects of 3-methylindole on specific tryptophan binding to hepatic nuclear envelope receptor and discusses the possible implications thereof.

Animals↗
PubMed1983

Asymmetric distribution and temperature-dependent clustering of filipin-sterol complexes in the nuclear membrane of Ehrlich ascites tumor cells.

When Ehrlich ascites tumor (EAT) cells were fixed at 4 degrees C and freeze-fractured, patchy areas having no intramembrane particles were visible in the nuclear envelope. The particle free areas (PFAs) were not seen on fixation at 28 degrees C, indicating that appearance of PFAs was caused by a kind of thermotropic phase separation. The PFAs were detected only in the nuclear membrane, and not in the plasma membrane. Most of them were present in the outer membrane of the nuclear envelope. In cells fixed at 4 degrees C, and treated with filipin all the filipin-sterol complexes appeared in clusters located in the PFAs. In contrast, the filipin-sterol complexes were evenly distributed in cells fixed at 28 degrees C. This suggests that at low temperature, molecules of cholesterol gather in the PFAs. Temperature-dependent cluster formation was seen only in the complexes of the nuclear membrane, suggesting that the cholesterol in the nuclear membrane is more mobile than that in the plasma membrane. In addition, the distribution of filipin-sterol complexes in the nuclear envelope was asymmetric. The complexes were seen only in the outer (cytoplasmic), but not in the inner (nucleoplasmic) membrane of the nuclear envelope, reflecting differences in the structural, and presumably functional, characteristics of the outer and inner nuclear membranes.

Animals↗
PubMed1996

Description of primordial germ cells, oogonia, oocytes and embryo-like growth in squash preparations of tissues from hematological malignancies.

This study evidences the presence of primordial germ cells, in tissue squash preparations and sections from hematological malignancies. Primordial germ cells were identified by their morphology, the intense PAS, PAS-D reaction and presence of calcium-activated neutral proteinase. Primordial germ cells gave rise to nuclear vlimata. Immature oogonia exhibited a nuclear envelope and a star-shaped nuclear core, arising from acellular globose bodies impregnated by a nuclear vlima of primordial germ cell. Bone marrow tissue oogonia were PAS and PAS-D positive, identical to fungal ones. Calcium-activated neutral proteinase was demonstrated in the plasma of the acellular globose bodies, the nuclear envelope and the conglomerated primordial germ cells. Immature bone marrow oogonia progressed into mature ones, leptotene, diplotene, dictyotene and mature oocytes. Nuclear vlimata fertilized primordial germ cells, oogonia and oocytes, giving rise to round embryos at the morula and hatching morula-like stages. Embryos consisted of a zonapellucida-like cortex, composed of glycosaminoglycans, glycoproteins, protease and diffuse nuclear material, enclosing developing cells. Primordial germ cells, oogonia and embryos were also demonstrated in squash preparations of adult rat testis and sections of normal rat bone marrow tissues. The observations document that primordial germ cells are the primary stem cells which give rise to nuclear vlimata and oogonia, which constitute the secondary stem germ cells. The results are discussed in terms of stem cell renewal according to the events: primordial germ cells-gametes-fertilization-embryos-primordial germ cells.

Animals↗
PubMed1978

Mitosis in the hemoflagellate Trypanosoma cyclops.

The ultrastructure of interphase and mitotic nuclei of the epimastigote form of Trypanosoma cyclops Weinman is described. In the interphase nucleus the nucleolus is located centrally while at the periphery of the nucleus condensed chromatin is in contact with the nuclear envelope. The nucleolus fragments at the onset of mitosis, but granular material of presumptive nucleolar origin is often recognizable in the mitotic nucleus. Peripheral chromatin is in contact with the nuclear envelope throughout mitosis, and it seems reasonable to assume that the nuclear envelope is involved in its segregation to the daughter nuclei. Spindle microtubules extend between the poles of the dividing nucleus and terminate close to the nuclear envelope. The basal body and kinetoplast divide before the onset of mitosis and do not appear to have any morphologic involvement in that process. Spindle pole bodies, kinetochores, and chromosomal microtubules have not been observed.

Animals↗
PubMed1997

Heterogeneous behavior of cells with respect to induction of retrograde transport from the trans-Golgi network to the Golgi upon inhibition of the vacuolar proton pump.

We investigated whether the vacuolar proton pump is required for proper vesicular trafficking through the trans-Golgi network. Previously, we provided evidence that endocytic transport to early and late endosomes, as well as recycling from endosomes to the plasma membrane, but not transport to lysosomes, continued in the presence of bafilomycin A1, a specific inhibitor of the vacuolar proton pump (Van Weert et al., J. Cell Biol. 130, 821-834 (1995)). Following endocytosis, recycling of cell surface glycoproteins occurs directly from endosomes to the plasma membrane, but also involves trafficking via the trans-Golgi network. We now used transferrin conjugated to horseradish peroxidase (Tf/HRP) to label and monitor the itinerary of the transferrin receptor using electron microscopy. In the presence of bafilomycin A1, transport of Tf/HRP to the trans-Golgi network continued. However, in a limited percentage of cells bafilomycin A1 induced transport of Tf/HRP into the Golgi stack. Retrograde transport into the Golgi stack was analyzed further in cells that were additionally treated with Brefeldin A, a drug that induces a redistribution of Golgi, but not trans-Golgi network markers, to the endoplasmic reticulum and the nuclear envelope. Sequential addition of bafilomycin A1 and Brefeldin A to exponentially growing A431 cells resulted in heavy Tf/HRP-labeling of the endoplasmic reticulum and the nuclear envelope in about 3% of the cells. No Tf/HRP was detected in endoplasmic reticulum or nuclear envelope of the other cells. the same observation was made for HepG2, CHO, and HeLa cells or when HRP was used as a fluid phase-endocytosed marker. Experiments performed with cell cycle-synchronized A431 cells revealed that retrograde transport was not linked to any specific phase of the cell cycle. The presence of epidermal growth factor, however, increased the amount of positive nuclear envelopes in A431 cells to 8%. In conclusion, our results show that inhibition of the vacuolar proton pump by bafilomycin A1 induces retrograde transport from the trans-Golgi network into the Golgi, and indicate that clonal cell lines display heterogeneity in this respect.

Anti-Bacterial Agents↗
PubMed2003

RanGTP mediates nuclear pore complex assembly.

In metazoa, the nuclear envelope breaks down and reforms during each cell cycle. Nuclear pore complexes (NPCs), which serve as channels for transport between the nucleus and cytoplasm, assemble into the reforming nuclear envelope in a sequential process involving association of a subset of NPC proteins, nucleoporins, with chromatin followed by the formation of a closed nuclear envelope fenestrated by NPCs. How chromatin recruitment of nucleoporins and NPC assembly are regulated is unknown. Here we demonstrate that RanGTP production is required to dissociate nucleoporins Nup107, Nup153 and Nup358 from Importin beta, to target them to chromatin and to induce association between separate NPC subcomplexes. Additionally, either an excess of RanGTP or removal of Importin beta induces formation of NPC-containing membrane structures--annulate lamellae--both in vitro in the absence of chromatin and in vivo. Annulate lamellae formation is strongly and specifically inhibited by an excess of Importin beta. The data demonstrate that RanGTP triggers distinct steps of NPC assembly, and suggest a mechanism for the spatial restriction of NPC assembly to the surface of chromatin.

Amino Acid Substitution↗
PubMed1995

GTP hydrolysis by Ran occurs at the nuclear pore complex in an early step of protein import.

Mediated import of proteins into the nucleus involves multiple cytosolic factors, including the small GTPase Ran. Whether Ran functions by interacting with other cytosolic proteins or components of the nuclear pore complex has been unclear. Furthermore, the precise transport step where Ran acts has not been determined. To address these questions, we have analyzed the binding interactions of Ran using permeabilized cells and isolated nuclear envelopes. By light and electron microscope immunolocalization, we have found that Ran accumulates specifically at the cytoplasmic surface of the nuclear pore complex when nuclear import in permeabilized cells is inhibited by nonhydrolyzable analogs of GTP. Ran associates with a peripheral pore complex region that is similar to the area where transport ligands accumulate by depletion of ATP, which arrests an early step of transport. Binding studies with isolated nuclear envelopes in the absence of added cytosol indicate that Ran-GTP directly interacts with a pore complex protein. Using blot overlay techniques, we detected a single prominent polypeptide of isolated nuclear envelopes that binds Ran-GTP. This corresponds to the 358-kD protein RanBP2, a Ran binding pore complex protein recently identified by two-hybrid screening. Thus, RanBP2 is likely to constitute the Ran-GTP-binding site detected at the cytoplasmic periphery of the pore complex. These data support a model in which initial ligand binding to the nuclear pore complex occurs at or near RanBP2, and that hydrolysis of GTP by Ran at this site serves to define commitment to the nuclear import pathway.

Adenosine Triphosphate↗