Assembly of 5S ribosomal RNA is required at a specific step of the pre-rRNA processing pathway.

A collection of yeast strains surviving with mutant 5S RNA has been constructed. The mutant strains presented alterations of the nucleolar structure, with less granular component, and a delocalization of the 25S rRNA throughout the nucleoplasm. The 5S RNA mutations affected helix I and resulted in decreased amounts of stable 5S RNA and of the ribosomal 60S subunits. The shortage of 60S subunits was due to a specific defect in the processing of the 27SB precursor RNA that gives rise to the mature 25S and 5.8S rRNA. The processing rate of the 27SB pre-rRNA was specifically delayed, whereas the 27SA and 20S pre-rRNA were processed at a normal rate. The defect was partially corrected by increasing the amount of mutant 5S RNA. We propose that the 5S RNA is recruited by the pre-60S particle and that its recruitment is necessary for the efficient processing of the 27SB RNA precursor. Such a mechanism could ensure that all newly formed mature 60S subunits contain stoichiometric amounts of the three rRNA components.

Ultrastructural localization of rRNA shows defective nuclear export of preribosomes in mutants of the Nup82p complex.

To study the nuclear export of preribosomes, ribosomal RNAs were detected by in situ hybridization using fluorescence and EM, in the yeast Saccharomyces cerevisiae. In wild-type cells, semiquantitative analysis shows that the distributions of pre-40S and pre-60S particles in the nucleolus and the nucleoplasm are distinct, indicating uncoordinated transport of the two subunits within the nucleus. In cells defective for the activity of the GTPase Gsp1p/Ran, ribosomal precursors accumulate in the whole nucleus. This phenotype is reproduced with pre-60S particles in cells defective in pre-rRNA processing, whereas pre-40S particles only accumulate in the nucleolus, suggesting a tight control of the exit of the small subunit from the nucleolus. Examination of nucleoporin mutants reveals that preribosome nuclear export requires the Nup82p-Nup159p-Nsp1p complex. In contrast, mutations in the nucleoporins forming the Nup84p complex yield very mild or no nuclear accumulation of preribosome. Interestingly, domains of Nup159p required for mRNP trafficking are not necessary for preribosome export. Furthermore, the RNA helicase Dbp5p and the protein Gle1p, which interact with Nup159p and are involved in mRNP trafficking, are dispensable for ribosomal transport. Thus, the Nup82p-Nup159p-Nsp1p nucleoporin complex is part of the nuclear export pathways of preribosomes and mRNPs, but with distinct functions in these two processes.

Assembly and functional organization of the nucleolus: ultrastructural analysis of Saccharomyces cerevisiae mutants.

Using Saccharomyces cerevisiae strains with genetically modified nucleoli, we show here that changing parameters as critical as the tandem organization of the ribosomal genes and the polymerase transcribing rDNA, although profoundly modifying the position and the shape of the nucleolus, only partially alter its functional subcompartmentation. High-resolution morphology achieved by cryofixation, together with ultrastructural localization of nucleolar proteins and rRNA, reveals that the nucleolar structure, arising upon transcription of rDNA from plasmids by RNA polymerase I, is still divided in functional subcompartments like the wild-type nucleolus. rRNA maturation is restricted to a fibrillar component, reminiscent of the dense fibrillar component in wild-type cells; a granular component is also present, whereas no fibrillar center can be distinguished, which directly links this latter substructure to rDNA chromosomal organization. Although morphologically different, the mininucleoli observed in cells transcribing rDNA with RNA polymerase II also contain a fibrillar subregion of analogous function, in addition to a dense core of unknown nature. Upon repression of rDNA transcription in this strain or in an RNA polymerase I thermosensitive mutant, the nucleolar structure falls apart (in a reversible manner), and nucleolar constituents partially relocate to the nucleoplasm, indicating that rRNA is a primary determinant for the assembly of the nucleolus.

The role of the Schizosaccharomyces pombe gar2 protein in nucleolar structure and function depends on the concerted action of its highly charged N terminus and its RNA-binding domains.

Nonribosomal nucleolar protein gar2 is required for 18S rRNA and 40S ribosomal subunit production in Schizosaccharomyces pombe. We have investigated the consequences of the absence of each structural domain of gar2 on cell growth, 18S rRNA production, and nucleolar structure. Deletion of gar2 RNA-binding domains (RBDs) causes stronger inhibition of growth and 18S rRNA accumulation than the absence of the whole protein, suggesting that other factors may be titrated by its remaining N-terminal basic/acidic serine-rich domain. These drastic functional defects correlate with striking nucleolar hypertrophy. Point mutations in the conserved RNP1 motifs of gar2 RBDs supposed to inhibit RNA-protein interactions are sufficient to induce severe nucleolar modifications but only in the presence of the N-terminal domain of the protein. Gar2 and its mutants also distribute differently in glycerol gradients: gar2 lacking its RBDs is found either free or assembled into significantly larger complexes than the wild-type protein. We propose that gar2 helps the assembly on rRNA of factors necessary for 40S subunit synthesis by providing a physical link between them. These factors may be recruited by the N-terminal domain of gar2 and may not be released if interaction of gar2 with rRNA is impaired.

Influence of 12-O-tetradecanoylphorbol-13-acetate on proliferation and maturation of human breast carcinoma cells (MCF-7): relationship to cell cycle events.

Exposure of MCF-7 human mammary carcinoma cells to 12-O-tetradecanoylphorbol-13-acetate (TPA) results in changes in cell morphology and arrest of cell growth. The inhibition of cell proliferation and the increase in cell volume are concentration dependent; these effects are reversible upon removal of the tumor promoting agent. Electron microscopic studies reveal that TPA increases endoplasmic reticulum and induces the appearance of secretory granules. MCF-7 cells treated by TPA therefore present morphological characteristics of secretory cells. These effects of TPA on MCF-7 cells are accompanied by specific disruption of cell cycle events, a block of cells in G1 at the expense of S base, and a delayed passage through G2. Studies in which a cell cycle lock in G1 is produced by tamoxifen show that exposure of such cells to PA produces cell morphological changes and an inability to progress through the cell cycle when estradiol is added.

Reversible plasma membrane ultrastructural changes correlated with electropermeabilization in Chinese hamster ovary cells.

Chinese hamster ovary cells (CHO) grown in monolayers were permeabilized to molecules with molecular weight up to 1000 by high intensity 100 mus square wave electric field pulses. This permeability was transient and the cell viability was not affected. It was not possible for molecules with molecular weight larger than 1500 to penetrate inside the cytoplasm if lytic pulsing conditions were not used. In order to investigate the ultrastructural changes associated with this transient and limited permeabilization, cells were chemically fixed a few seconds after their pulsation and observed by electron microscopy. By scanning electron microscopy, numerous microvilli and blebs were observed almost immediately after application of the field. No other membrane changes were observed. Permeabilization of the membrane was visualized at the electron microscopic level by penetration of Ruthenium red. The appearance of osmotic pressure-dependent 'blebs' was indicative of local weakening of the plasma membrane. Most of these effects were fully reversible and disappeared within 30 min at 37 degrees C with the formation of huge polykaryons when cells were in contact before pulsing.

Basic fibroblast growth factor (FGF-2) internalization through the heparan sulfate proteoglycans-mediated pathway: an ultrastructural approach.

Biochemical studies have shown that basic fibroblast growth factor (bFGF or FGF-2) is internalized by two pathways, after binding to either FGF tyrosine kinase receptors or to heparan sulfate proteoglycans (HSPG). To get insights on the HSPG-mediated pathway, we have examined by electron microscopy the intracellular route of bFGF-HRP, a monovalent conjugate of bFGF and horseradish peroxidase which was found to bind to HSPG only and was detectable by electron microscopy. bFGF-HRP association to adult bovine aortic endothelial (ABAE) cells or baby hamster kidney (BHK) cells was inhibited by a high molar excess of native bFGF, a 2 M NaCl wash at neutral pH, heparin and heparan sulfate, but not by chondroitin 4-sulfate or chondroitin 6-sulfate. bFGF-HRP was not able to displace [125I]bFGF from its high-affinity binding sites, and the dissociation constant of its binding to ABAE cells was estimated at 3 nM. Time-course experiments were performed to follow bFGF-HRP endocytosis in ABAE cells. bFGF-HRP was found to enter the cell after binding to the plasma membrane or extracellular matrix. On the cell surface, the probe accumulated in noncoated flask-shaped invaginations and in caveolae rather than in clathrin-coated pits. Immediately after endocytosis, bFGF-HRP was detected in pleiomorphic tubulovesicular and tubulocisternal early endosomes. Multivesicular bodies contained diaminobenzidine (DAB) precipitate after 5 to 15 min, but lysosomes were not labeled before 1 h, indicating a delayed transfer from late endosomes to lysosomes. Labeling was never detected in the nucleus, even after intensification of the DAB reaction product by silver-gold enhancement. Similar endocytic pathways and intracellular locations were observed in other endothelial and non-endothelial cell types. These results suggest that bFGF associated to HSPG can enter the cell via several pathways and follows mainly a degradative route.

Basic fibroblast growth factor (FGF-2) is addressed to caveolae after binding to the plasma membrane of BHK cells.

bFGF endocytosis in BHK cells was examined by electron microscopy using a conjugate of recombinant human bFGF and digoxigenin (bFGF-DIG). This probe keeps the biological activity of non-labeled bFGF and can be readily detected with anti-digoxigenin antibodies (Gleizes et al., Anal. Biochem. 219, 360-367 (1994)). Time-course studies of bFGF-DIG endocytosis were performed by incubating BHK cells at 4 degrees C in the presence of first 20 ng/ml bFGF-DIG and then antidigoxigenin antibodies adsorbed onto 10-nm gold particles. A semi-quantitative study revealed that caveolae were the main endocytic pathway of bFGF-DIG in these cells, whereas clathrin-coated pits were scarcely labeled. After occurring in caveolae, bFGF-DIG was sequentially detected in tubulovesicular early endosomes, multivesicular late endosomes, and lysosomes. Under the same conditions, low density lipoprotein (LDL)-gold was seen entering the cell exclusively through clathrin-coated pits. However, LDL-gold and bFGF-DIG were colocalized, at least in part, in common endosomal structures. Pretreatment of the cells with phosphatidylinositol-phospholipase C reduced the proportion of membrane-bound bFGF-DIG in caveolae, but did not inhibit bFGF-DIG presence in caveolae. These data suggest that bFGF enters into BHK cells through caveolae and is then shuttled into a degradative pathway similar to that of LDL.

Structural and functional analysis of the nucleolus of the fission yeast Schizosaccharomyces pombe.

Yeasts are an attractive model for the study of ribosome synthesis. However, our understanding of the relationship between the structure and function of the yeast nucleolus, in which preribosomal particles are synthesized, requires further investigations using microscopic approaches and in situ molecular biology. Combining cryofixation and cryosubstitution of Schizosaccharomyces pombe, we could identify morphologically distinct substructures in the nucleolus similar to the components of nucleoli of higher eukaryotes such as the fibrillar centers (FCs), the dense fibrillar component (DFC) and the granular component (GC). We complemented this morphological study by performing in situ hybridization and immunocytochemistry at the electron microscopy level. Using a probe complementary to the entire rRNA transcription unit of S. pombe, we detected rDNA at the periphery of the FCs, while immunocytochemistry with antibodies specific for the RNA polymerase I and the gar1 protein provided evidence that transcription and early steps of maturation take place in the DFC that extends throughout the nucleolus. We also present evidence that preribosomal subunits may be exported along tracks to the cytoplasm through all of the pores of the nuclear envelope and not just those in the portion of the envelope close to the nucleolus.

Cerium-based ultracytochemical localization of aspartate transcarbamylase activity in the cell membrane complex of Saccharomyces cerevisiae.

Aspartate transcarbamylase (ATCase) activity was localized ultracytochemically in the yeast Saccharomyces cerevisiae by precipitation of its reaction product orthophosphate as cerium phosphate. We prefixed yeast cells with ice-cold 1% glutaraldehyde for 30 min which preserved 80% of ATCase activity. Cells were washed and incubated with ATCase substrates (aspartate, carbamyl phosphate) plus cerium chloride, and postfixed by osmium tetroxide. In cells from exponential batch cultures, deposits of cerium phosphate delineated simultaneously or alternatively membranes of the secretory pathway: nuclear envelope, endoplasmic reticulum, Golgi complex and the plasmalemma; mitochondrial membranes and intramitochondrial fibrous component were labelled as well. Deposits of cerium phosphate were never observed in the nucleoplasm. Cells incubated in the absence of cerium or ATCase substrates and mutant S. cerevisiae cells lacking ATCase activity served as controls. Small round electron-dense condensates were found to be randomly distributed within some cells, both in control and experimental runs, in the nucleoplasm, cytoplasm and mitochondrial matrix and represented undefined osmicated endogenous compounds. Our results suggest that the synthesis of pyrimidine precursors occurs in membranes, where compounds such as UDP-glucose and CDP-diglycerides are needed for membrane and/or yeast cell wall synthesis. The possible contribution of ATCase activity found in the nuclear envelope to nucleic acid synthesis remains to be clarified.

Electron microscopic study on gut epithelium of the tench (Tinca tinca L.) with respect to its absorptive functions.

Few morphological differences are seen along the intestinal tract of the adult tench (Tinca tinca L.) a stomachless freshwater teleost. However, three segments can be distinguished, when function and structure of enterocytes are studied. The enterocytes of the proximal segment are found to be concerned with dietary lipids absorption. In the cell, absorbed fats are seen in two inclusion bodies: lipid particles and lipid droplets. Only lipid particles are involved in direct transport of absorbed fatty acids in the blood circulation, as in lymphatic vessels. Lipid droplets seem to be involved in temporary storage of fatty acids. Special features are found in enterocytes of the short middle segment; these cells show many invaginations and pinocytosis figures, a well-developed tubulo-vesicular network and large vacuoles in the supranuclear hyaloplasm. Such characters bear a resemblance to descriptions of the gut of some newborn mammals. The great permeability of this epithelium to macromolecules is demonstrated by the administration of horseradish peroxidase (HRP). Enterocytes of the distal segment show, at their basal pole, numerous invaginations of the plasma membrane, and a large mitochondrial population. Morphological similarity suggests a functional analogy with epithelia involved in water and ions transport.

Structure and function of the intestinal epithelial cells in the perch (Perca fluviatillis L.).

An ultrastructural study of the intestinal absorptive epithelium in perch (Perca fluviatilis) has shown that the perch intestine can be divided into three segments: the proximal segment, the middle segment and the distal segment. The enterocytes of the proximal segment are found to be concerned with lipid absorption. The adsorbed fat gives rise to the presence of two forms of inclusions: lipid particles and lipid droplets. Enterocytes of the middle segment exhibit the typical ultrastructural features of pinocytosis; these consist of extensive invaginations of the luminal surface membrane and acculation of vacuoles in the apical cytoplasm. Exogenous proteins are ingested by absorptive cells from the intestinal lumen by a process similar to that described in neonatal mammals. In the distal segment the absorptive cells have few, short microvilli. Besides the absorptive epithelial cells, goblet cells, endocrine cells, pear-shaped cells, and plasma cells are occasionally found.

Ultrastructure of endocrine cells in the stomach of two teleost fish, Perca fluviatilis L. and Ameiurus nebulosus L.

In the gastric mucosa of two teleost species, the perch (Perca fluviatilis) and the catfish (Ameiurus nebulosus) three endocrine cell types were found, located predominantly between the mucoid cells of the gastric mucosa. A fourth cell type is present in the gastric glands of catfish. Each cell type was defined by its characteristic secretory granules. Type-I cells were predominant in both fish. These cells contained round or oval granules with a pleomorphic core. The average diameter of granules was 400 nm for the perch and 270 nm for the catfish. Type-II cells of both species displayed small, highly osmiophilic granules about 100 nm in diameter. The secretory granules of type-III cells (260 nm in the perch and 190 nm in the catfish) were round or slightly oval in shape and were filled with a finely particulate electron-dense material. Type-IV cells of the catfish were found in the gastric glands only. Their cytoplasm was filled with homogeneous, moderately electron-dense granules averaging 340 nm in diameter. The physiological significance of these different morphological types of gastric endocrine cells requires further investigation.

Labeling of basic fibroblast growth factor with digoxigenin: a nonradioactive probe for biochemical and cytological applications.

Digoxigenin, a 391-Da plant sterol, was conjugated to recombinant bFGF with the aim of detecting it with high specificity and sensitivity in cultured eukaryotic cells using antibodies against digoxigenin. The conjugate, bFGF-DIG, displayed a mitogenic activity on endothelial cells equivalent to that of nonlabeled bFGF. Binding of the probe on the cell surface was assessed by ELISA on cells, which allowed discrimination between low- and high-affinity bFGF binding sites. Using a chemiluminescent system, chemical cross-linking of bFGF-DIG with FGF receptors was analyzed directly on Western blots of cell extracts with anti-digoxigenin antibodies. The labeling pattern was identical to that reported with iodinated bFGF, showing that bFGF-DIG bound to the same receptors. The time course of intracellular degradation of internalized bFGF-DIG was also followed by immunodetection on Western blots: the low speed of the catabolic process and the size of the degradation products were comparable to those previously described with iodinated bFGF. In parallel, bFGF-DIG was readily detected by immunofluorescence in cultured cells, and was shown to be an interesting probe to determine bFGF endocytosis pathways by electron microscopy. bFGF-DIG appeared as a multifunctional nonradioactive probe suitable for combined biochemical and cytological studies of bFGF.

Location of ribosomal genes in CHO cells; in situ hybridization with a non-isotopic DNA probe on G-banded chromosomes.

A novel in situ hybridization technique using sulfonated probes is described. This non-radioactive approach, which employs chemically modified DNA and immunocytochemical procedures, is compatible with pre-G-banding and allows a rapid localization of the hybridized sequences on chromosomal spreads with a high spatial resolution. Using this technique we have localised the Chinese hamster ribosomal genes in the telomeric region of ten chromosomes, and among them in the subtelomeric q region of the Z5 chromosome. These results are discussed, the genetic markers confirming and locating the origin of Z group chromosomes by rearrangements of Chinese hamster chromosomes.

Immunolocalization of the 100 kDa nucleolar protein during the mitotic cycle in CHO cells.

The localization of a major nucleolar protein with a molecular weight of 100,000 has been followed during mitosis in Chinese hamster ovary CHO cells using specific antibodies to this protein and immunocytochemical techniques. The 100 kDa protein was visualized at discrete sites on metaphase chromosomes, corresponding to nucleolus organizer regions, and in large, immunostained nucleolar remnants that are discarded in the cytoplasm after nucleolar disintegration. After mitosis, the 100 kDa protein was shown to play an early role in nucleolar reformation. It was first detected in small deposits around the anaphase chromosomes. In telophase, the protein accumulated simultaneously in prenucleolar bodies and in the reforming nucleoli. The early presence of the 100 kDa protein in the telophase nucleus suggests that it is essential for the reestablishment of nucleolar function after mitosis. Thus this protein is present throughout the CHO cell cycle, an observation which supports the hypothesis that it plays a fundamental role in cell organization.

Bismuth staining of a nucleolar protein.

A major nucleolar protein in Chinese hamster ovary cells with a molecular weight (MW) of 100 kD has been found to stain selectively with the bismuth tartrate technique of Locke & Huie [19]. After glutaraldehyde fixation and bismuth staining of electrophoretic transfers of total nucleolar proteins separated by SDS-PAGE, a single band corresponding to the 100 kD protein is revealed. When the technique is applied to whole cells, small punctate regions of the nucleoli are strongly stained. At the ultrastructural level, bismuth selectively contrasts the fibrillar centers and the adjoining cords of the dense fibrillar component. The remainder of the dense fibrillar component is not stained. It is proposed that the high phosphorylation level of the 100 kD protein is responsible for its glutaraldehyde-insensitive bismuth staining. The concentration of this protein in certain localized regions of the nucleolus suggests that it plays a metabolic rather than a structural role.

Immunolocalization of the 100 K nucleolar protein in CHO cells.

The localization of a major nucleolar protein with a molecular weight of 100,000 has been followed in Chinese hamster ovary cells using specific antibodies to this protein and immunocytochemical techniques. By immunofluorescence and immunocytochemistry at ultrastructural level in situ, the 100 K protein was detected abundantly in the nucleolus of interphase cells. In exponentially growing cells, the dense fibrillar component was shown to contain more 100 K protein than the granular RNP component but both the nucleolar components were positively immunostained. Fibrillar centers consistently showed weaker or no staining. The 100 K association with the preribosomal RNP components of the interphase nucleolus supports evidence for a role of this protein in pre-rRNA transcription and preribosomal processing.

The effect of adenosine analogue (DRB) on a major nucleolar phosphoprotein nucleolin.

Nucleolin, a phosphorylated nucleolar protein, of 100 kDa selectively stained with bismuth tartrate and silver nitrate, is implicated in the transcription and maturation of pre-ribosomal RNA. Nucleolin also fulfills a structural function in nucleolar organization. Using immunocytochemistry the action of 5-6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB), an inhibitor of hn/RNA synthesis known to modify the organization of the nucleolus, was studied for its effects on the distribution and the amount of nucleolin present. After DRB treatment, the morphology of the nucleolus was rapidly disturbed, but the distribution of the nucleolin remained unchanged: the dense fibrillar and the granular components were always positively immunostained. Thirty min after incubation with the drug, a strong increase of the amount of nucleolin occurred. Prolonged treatment led to a marked loss of label. Silver and bismuth staining showed that DRB does not seem to significantly affect the phosphorylation of nucleolin.