Ultrastructural localization of RNA in cryptomonads

Summary In a previous study, DNA was localized in cells of two cryptomonads,Pyrenomonas sp. andCryptomonas ovata, by use of immuno-gold technique. Of particular interest was the ultrastructural localization of DNA in the nucleomorph, supposed to be a vestigial nucleus of a former endosymbiont [Hansmann Pet al. (1986) Eur J Cell Biol 42: 152–160]. In the present paper, distribution of RNA in the same two organisms is reported. RNA was detected by the specific and very sensitive RNase-gold method. RNA could be demonstrated in all of the four plasmatic compartments of cryptomonad cells (cytoplasm, periplastidal compartment, mitochondrion, and plastid), although the amounts differed greatly in the respective compartments. In the nucleus, the condensed chromatin and the nucleolus were preferentially labeled. Intense labeling could also be found over the fibrillogranular region of the nucleomorph. This fact lends strong support to the supposition that the fibrillogranular body represents the structural and functional equivalent of a nucleolus and thus again supports the hypothesis that the nucleomorph represents a vestigial eukaryotic nucleus. InPyrenomonas sp., gold-particle density over the nucleolus and the fibrillogranular body was quantitatively evaluated in order to compare their respective RNA synthesizing activities. Labeling density over the nucleolus was found to be 2.7 times higher and thus, on account of its greater volume, the nucleolus may contain 17 times more RNA than the fibrillogranular body of the nucleomorph.Abbreviations BSA bovine serum albumin - ER endoplasmic reticulum - GA glutaraldehyde - SSC standard saline citrate - SSCB SSC containing BSA     

Ultrastructural localization of nuclei acids by the use of enzyme-gold complexes

A cytochemical technique for the ultrastructural localization of substrates using enzyme-gold complexes is reported. RNase A and DNase I have been labeled with gold particles. The RNase-gold and dNase-gold complexes obtained were applied on thin sections of glutaraldehyde-fixed and Epon-embedded tissues. Different cellular compartments were labeled by these enzyme-gold complexes. Using the RNase-gold complex the rough endoplasmic reticulum appeared decorated with gold particles. The gold marker was also present over the nucleus, especially over the nucleolus; mitochondria were weakly labeled. Using the DNase-gold complex, gold particles were concentrated over the euchromatin of the nucleus and the mitochondria. The heterochromatin and the nucleolus showed a less intense labeling. For both enzyme-gold complexes, the Golgi area, the secretory granules and the extracellular space appeared free of label. In those control conditions where the substrates were added to the enzyme-gold complexes a major reduction in the labeling was observed. A quantitative evaluation of the labeling was performed. This evaluation confirmed the qualitative observations and the marked reduction of labeling occurring under the control conditions. The combination of the specificity of the enzyme-substrate interactions with the size and electron density of the gold particles and the good ultrastructural preservation of the tissues resulted in a very specific labeling with high resolution. These results demonstrate the possibility of detecting substrates by means of enzyme-gold complexes at the electron microscope level.     

不同生殖期鳜肝脏超微结构变化的观察

应用透射电镜对生殖季节与非生殖季节鳜肝脏超微结构的变化进行了观察。鳜肝细胞含有单个卵圆形的核,核仁清楚;细胞质内含有粗面内质网、线粒体、糖原颗粒和脂滴等细胞器和内含物。胆小管由相邻的数个肝细胞质膜凹陷围成,而肝血窦则由内皮细胞的胞质构成。还发现了贮脂细胞、枯否氏细胞和成纤维细胞。胆小管腔和窦周隙内浸润许多由肝细胞发出的微绒毛结构。鳜肝细胞的超微结构在产卵前后呈现明显变化：产卵前的肝细胞内富含线粒体、糖原颗粒和脂滴,粗面内质网发达;而产卵后的肝细胞内核仁发生迁移,部分细胞核囊泡化,糖原颗粒和脂滴排空,少数肝细胞具双核结构。非生殖期多数肝细胞核含有双核仁结构,胞质内溶酶体数量增多。     

A correlated morphometric and cytochemical study on hepatocyte nucleolar size and RNA distribution during vitellogenesis

Summary The enzyme-gold cytochemical technique was used to label RNA in the nucleolus and rough endoplasmic reticulum (RER) of the hepatocytes of normal, male American bullfrogs (Rana catesbeiana) and in bullfrogs eight days following treatment with estradiol-17. Concurrently, stereology was applied to quantitate: (1) the density of RNA labelling, and (2) changes in the size of the nucleus and nucleolus in response to estrogen treatment. In the hepatocytes from untreated frogs, specific labelling for RNA was present over the fibrillar and, to a greater extent, the granular portions of the nucleolus, and, to the greatest extent, over the RER. Following estrogen treatment, the density of RNA labelling increased over both parts of the nucleolus but was unchanged over the RER. The size of the nucleolus enlarged in response to estrogen: in combination with the increase in its RNA labelling, this suggested an increase of about 80% in the total amount of RNA in the nucleolus. Previous data on enlargement of the RER compartment, along with the present data on RNA labelling of RER, suggested that the total amount of this nucleic acid increased about 430% in this entity, in response to estrogen. However, the density of RNA labelling over the RER appears to be constant in spite of changes in the amount of RER.     

A nuclear localization sequence endows human pancreatic ribonuclease with cytotoxic activity

Some members of the ribonuclease superfamily, such as Onconase, are cytotoxic to cancer cells. This is not the case for human pancreatic ribonuclease. This lack of cytotoxicity is probably a result of the inhibition exerted by the cytosolic ribonuclease inhibitor once the protein has reached the cytosol. Until now, all cytotoxic human pancreatic ribonuclease variants have been described as being resistant to the inhibitor. Here, we report on the characterization of a cytotoxic variant of human pancreatic ribonuclease which has an Arg triplet introduced onto one of its surface-exposed loops. Despite its sensitivity to the inhibitor, this variant, called PE5, was only 5-15 times less cytotoxic than Onconase. When it was taken up by cells, it was only observed within late compartments of the endocytic pathway, probably because the number of molecules transported to the cytosol was too small to allow their visualization. Nuclear import assays showed that the Arg triplet endows PE5 with a nuclear localization signal. In these experiments, PE5 was efficiently transported to the nucleus where it was initially localized in the nucleolus. Although the Arg introduction modified the net charge of the protein and somehow impaired recognition by the cytosolic inhibitor, control variants, which had the same number of charges or were not recognized by the inhibitor, were not toxic. We concluded that targeting a ribonuclease to the nucleus results in cytotoxicity. This effect is probably due to ribonuclease interference with rRNA processing and ribosome assembly within the nucleolus.     

Electron-radioautographic analysis of 3H-thymidine distribution in the cell nuclei of the Chinese hamster

Three types of the label localization in the nuclei of Chinese hamster fibroblasts, growing for 9 and 13 hours with 3H-thymidine, were detected using electron microscopic autoradiography: 1. The label is relatively evenly distributed throughout the karyoplasm. 2. Silver grains are concentrated as stripes through the nucleus; a high label density is also found in the nuclear periphery and around the nucleolus. 3. The label is mainly concentrated over the condensed chromatin adjacent to the nuclear membrane. The cells labeled in the first half of S-phase and selected with colchicine in postsynthetic phase of the 1st and the 2nd cycles are characterized by the second and third types of label distribution. In the cell nuclei fixed in the postsynthetic period of the second cycle, the label localization in stripes is discontinuous. The results indicate that during cell transition from S to G2 the newly-synthetized DNA changes its localization in the nucleus. It is suggested that the second type of label distribution depends on the interphase chromosome concentration in definite zones of the nuclear volume after S-phase termination, and the third type label localization is connected with the formation of prophase chromosomes.     

Cytochemical characterization of two types of nuclear bodies in Cicer arietinum L.

The present study was carried out to characterize two distinct types of nuclear bodies, the nucleolus-associated bodies (NABs) and the satellites (SATs) using some specific staining, enzyme and immunogold labeling techniques in Cicer arietinum L. These bodies are of interest as the functional components of plant nucleus. DNA-specific staining and labeling with anti-DNA, a monoclonal antibody, were employed to verify the presence of DNA in NABs as well as in SATs. The enzyme-gold labeling technique was used to compare the relative amounts of RNase-gold and protease-gold labeling over the NABs. In NABs, RNase-gold labeling was relatively low compared to the protease-gold labeling. Ag-NOR staining revealed a similar content of NOR-silver proteins in both NABs and granular zone of the nucleolus. The NABs do not contain any DNA as they show negative response to DNA-specific stains and also when incubated with anti-DNA, only few gold particles are found over these structures. The SATs, on the other hand, react positively with DNA-specific stains, and high labeling is recorded with anti-DNA along with the dense chromatin masses.     

Use of halogenated precursors for simultaneous DNA and RNA detection by means of immunoelectron and immunofluorescence microscopy.

We have developed a novel approach for in situ labeling and detection of nucleic acids in cultured cells. It is based on in vivo incorporation of chlorouridine (ClU) or iododeoxyuridine (IdU) into Chinese hamster ovary cells with the aim of labeling RNA and DNA, respectively. The halogenated nucleotides are immunolabeled on ultrathin sections with anti-bromodeoxyuridine (BrdU) monoclonal antibodies that specifically react with either IdU or ClU. Furthermore, we combined ClU and IdU incubation to label simultaneously RNA and DNA in the same cell. Both were visualized by means of anti-BrdU antibodies exhibiting strong affinity for one of the two halogenated epitopes. Confocal imaging of interphase nuclei and electron microscopic analysis showed evidence of a partial colocalization of newly synthesized DNA and RNA inside the cell nucleus. RNase and DNase digestion of ultrathin sections after formaldehyde fixation and acrylic resin embedding confirmed the specificity of incorporation. Consequently, this method allows us to differentially label DNA and RNA on the same section. Using short pulses with the precursors, we could show that newly synthesized DNA and RNA both preferentially occur within the perichromatin region at the border of condensed chromatin domains.     

CYTOPLASMIC SYNTHESIS OF NUCLEAR PROTEINS : Kinetics of Accumulation of Radioactive Proteins in Various Cell Fractions after Brief Pulses

The synthesis of cytoplasmic and nuclear proteins has been studied in HeLa cells by examining the amount of radioactive protein appearing in the various subcellular fractions after labeling for brief periods. Due to the rapid equilibration of the amino acid pool, the total radioactivity in cytoplasmic protein increases linearly. The radioactivity observed in the cytoplasm is the sum of two components, the nascent proteins on the ribosomes and the completed proteins. At very short labeling times the specific activity of newly formed proteins found in the soluble supernatant fraction (completed protein) increases as the square of time, whereas the specific activity of the ribosomal fraction (nascent protein) reaches a plateau after 100 sec. The kinetics of accumulation of radioactive protein in the nucleus and the nucleolus is very similar to that of completed cytoplasmic protein, which suggests that the proteins are of similar origin. The rate of release and migration of proteins from the ribosomes into the nucleus requires less time than the synthesis of a polypeptide, which is about 80 sec. The uptake of label into nucleolar proteins is as rapid as the uptake of label into proteins of the soluble fraction of the cytoplasm, while nuclear proteins, including histones, tend to be labeled more slowly. The same results are obtained if protein synthesis is slowed with low concentrations of cycloheximide. The kinetics of incorporation of amino acids into various fractions of the cell indicates that the nucleus and the nucleolus contain few if any growing polypeptide chains, and thus do not synthesize their own proteins.     

p63cdc13, a B-type cyclin, is associated with both the nucleolar and chromatin domains of the fission yeast nucleus.

The cellular distribution of the fission yeast mitotic cyclin B, p63cdc13, was investigated by a combination of indirect immunofluorescence light microscopy, immunogold electron microscopy, and nuclear isolation and fractionation. Immunofluorescence microscopy of wild-type cells and the cold-sensitive mutant dis2.11 with a monospecific anti-p63cdc13 antiserum was consistent with the association of a major subpopulation of fission yeast M-phase protein kinase with the nucleolus. Immunogold electron microscopy of freeze-substituted wild-type cells identified two nuclear populations of p63cdc13, one associated with the nucleolus, the other with the chromatin domain. To investigate the cell cycle regulation of nuclear labeling, the mutant cdc25.22 was synchronized through mitosis by temperature arrest and release. Immunogold labeling of cells arrested at G2M revealed gold particles present abundantly over the nucleolus and less densely over the chromatin region of the nucleus. Small vesicles around the nucleus were also labeled by anti-p63cdc13, but few gold particles were detected over the cytoplasm. Labeling of all cell compartments declined to zero through mitosis. Cell fractionation confirmed that p63cdc13 was substantially enriched in both isolated nuclei and in a fraction containing small vesicles and organelles. p63cdc13 was not extracted from nuclei by treatment with RNase A, Nonidet P40 (NP-40), Triton X-100, and 0.1 M NaCl, although partial solubilization was observed with DNase I and 1 M NaCl. A known nucleolar protein NOP1, partitioned in a similar manner to p63cdc13, as did p34cdc2, the other subunit of the M-phase protein kinase. We conclude that a major subpopulation of the fission yeast mitotic cyclin B is targeted to structural elements of the nucleus and nucleolus.     

Nuclear localization of nucleocapsid-like particles and HCV core protein in hepatocytes of a chronically HCV-infected patient

Little is known about the life cycle of hepatitis C virus. Determination of the subcellular localization of HCV proteins may contribute to our understanding of the in vivo functions of the viral proteins. HCV core protein regulates multiple functions in host cells and it has been detected both in the cytoplasm and in the nucleus using different expression systems. In this study, nucleocapsid-like particles were observed in the nucleus of hepatocytes from a chronically HCV-infected patient. They were similar in size and shape to those of HCV core-like particles purified from recombinant Pichia pastoris cells. In addition the HCV core protein was detected not only in the cytoplasm but also in the nucleus and nucleolus of hepatocytes by immunoelectron microscopy. This is the first report showing nuclear localization of HCV core protein and nucleocapsid-like particles in hepatocytes during in vivo HCV infection.     

Labeling of hepatic glycogen after short- and long-term stimulation of glycogen synthesis in rats injected with 3H-galactose

The effects of short- and long-term stimulation of glycogen synthesis elicited by dexamethasone were studied by light (LM) and electron (EM) microscopic radioautography (RAG) and biochemical analysis. Adrenalectomized rats were fasted overnight and pretreated for short- (3 hr) or long-term (14 hr) periods with dexamethasone prior to intravenous injection of tracer doses of 3H-galactose. Analysis of LM-RAGs from short-term rats revealed that about equal percentages (44%) of hepatocytes became heavily or lightly labeled 1 hr after labeling. The percentage of heavily labeled cells increased slightly 6 hr after labeling, and unlabeled glycogen became apparent in some hepatocytes. The percentage of heavily labeled cells had decreased somewhat 12 hr after labeling, and more unlabeled glycogen was evident. In the long-term rats 1 hr after labeling, a higher percentage of heavily labeled cells (76%) was observed compared to short-term rats, and most glycogen was labeled. In spite of the high amount of labeling seen initially, the percentage of heavily labeled hepatocytes had decreased considerably to 55% by 12 hr after injection; and sparsely labeled and unlabeled glycogen was prevalent. The EM-RAGs of both short- and long-term rats were similar. Silver grains were associated with glycogen patches 1 hr after labeling; 12 hr after labeling, the glycogen patches had enlarged; and label, where present, was dispersed over the enlarged glycogen clumps. Analysis of DPM/mg tissue corroborated the observed decrease in label 12 hr after administration in the long-term animals. The loss of label observed 12 hr after injection in the long-term pretreated rats suggests that turnover of glycogen occurred during this interval despite the net accumulation of glycogen that was visible morphologically and evident from biochemical measurement.     

Analysis of apoptosis by cytometry using TUNEL assay

Activation of endonucleases that cleave chromosomal DNA preferentially at internucleosomal sections is a hallmark of apoptosis. DNA fragmentation revealed by the presence of a multitude of DNA strand breaks, therefore, is considered to be the gold standard for identification apoptotic cells. Several variants of the methodology that is based on fluorochrome-labeling of 3'-OH termini of DNA strand breaks in situ with the use of exogenous terminal deoxynucleotidyl transferase (TdT), commonly defined as the TUNEL assay, have been developed by us. This Chapter describes the variant based on strand breaks labeling with Br-dUTP that is subsequently detected immunocytochemically with Br-dUAb. Compared with other TUNEL variants the Br-dU-labeling assay offers the greatest sensitivity in detecting DNA breaks. Described also are modifications of the protocol that allow one to use other than Br-dUTP fluorochrome-tagged deoxynucleotides to label DNA breaks. Concurrent staining of DNA with propidium or 4',6-diamidino-2-phenylindole (DAPI) and multiparameter analysis of cells by flow- or laser scanning cytometry enables one to correlate induction of apoptosis with the cell cycle phase.     

Distribution of rDNA in the nucleus of Giardia lamblia: detection by Ag-I silver stain.

Previous investigations have proved that diplomonads have primitive cell nuclei and lack a nucleolus. We determined the distribution of ribosomal DNA (rDNA) in diplomonad nuclei that lacked a nucleolus. Giardia lamblia was used as the experimental organism with Euglena gracilis as the control. The distribution of rDNA was demonstrated indirectly by the modified Ag-I silver technique that can indicate specifically the nucleolus organizing region (NOR) by both light and electron microscopy. In ultrathin sections of silver stained Euglena cells, all silver grains were concentrated in the fibrosa of the nucleolus, while no silver grains were found in the cytoplasm, nucleoplasm, condensed chromosomes or pars granulosa of the nucleus. In the silver stained Giardia cells, no nucleolus was found, but a few silver grains were scattered in the nucleus. This suggests that the rDNA of Giardia does not form an NOR-like structure and that its nucleus is in a primitive state.     

Ultrastructural Distribution of DNA within Plant Meristematic Cell Nucleoli during Activation and the Subsequent Inactivation by a Cold Stress

We have investigated the precise location of DNA within the meristematic cell nucleolus ofZea maysroot cells andPisum sativumcotyledonary buds, in the course of their activation and induced inactivation following a subsequent treatment at low temperature. For this purpose, we combined the acetylation method, providing an excellent distinction between the various nucleolar components, with thein situterminal deoxynucleotidyl transferase-immunogold technique, a highly sensitive method for detecting DNA at the ultrastructural level. In addition to the presence of DNA in the condensed chromatin associated with the nucleolus, we demonstrated that a significant label was detected in the nucleolus of quiescent cells in both plant models. Evident labels were also found in the dense fibrillar component of actived nucleoli. Whereas in inactivated nucleoli no significant label was observed within the dense fibrillar component, an intense label was seen over the large heterogeneous fibrillar centres only during inactivation. The granular component was never significantly labelled. These results appear to indicate that the DNA present in the dense fibrillar component of activated nucleoli withdraws from this structure during its inactivation and becomes incorporated in the large fibrillar centres. These observations suggest that in plant cells inactivation of rRNA genes is clearly accompanied by changes in the conformation of ribosomal chromatin.     

Vegetative nuclear structures and their mode of division inCyathus species

Vegetative nuclear division in the homokaryotic and dikaryotic hyphae ofCyathus olla Brodie,C. setosus Brodie andC. bulleri Brodie was investigated. In the homokaryotic hyphae a nucleolus develops within a globular condensed nucleus consisting of a folded up filament. As the nucleolus increases in size, the nucleus unfolds and can assume a ring, horseshoe or filament configuration. The filament duplicates and (usually when unwound from the nucleolus) divides longitudinally. Occasionally, strand separation occurs while the filament is wrapped in the form of a ring around the nucleolus. The daughter nuclei may condense before the next division. In the dikaryotic hyphae the same nuclear cycle occurs as in the homokaryons except that an extra nuclear condensation to the globular form can occur in both the clamp and tube nuclei. The division of these two nuclei is not always synchronous and, moreover, the stage of karyokinesis of the clamp nucleus is not closely synchronized with the formation of the clamp connection. A deeply stained granule is associated with the nucleus. Some granules can be observed to be connected to the nucleus by a faintly Feulgen positive thread-like structure but other granules are sessile. The granule or centriole-like body is thought to direct the nuclear unfolding process. It may divide prior to, or after nuclear division.     

Simultaneous photometry of the DNA and silver-grain count in liver cells labelled with 3H-thymidine or 3H-leucine

A modified method was proposed for reflected light simultaneous measurement of DNA content and of the silver grain number in the nucleus or cytoplasm of the same cell. Specimens-smears of isolated liver cells incorporated 3H-thymidine and 3H-leucine were prepared on coverslips and after processing were mounted on the slide glasses with smeared side facing downwards to avoid the influence of grains on DNA content measurements. To decrease the background, label measurements were carried out in polarized light. It was shown that the intensity of 3H-leucine incorporation in hepatocytes increases proportionally with cell ploidy degree.