Coiled bodies in nuclei from plant cells evolving from dormancy to proliferation

To characterise the coiled bodies in meristematic nuclei of Saccharum officinarum, immunofluorescence labelling with antibodies against components of the splicing (U2B' and Sm core protein B) and pre-rRNA processing (fibrillarin) complexes was used in cells from the dormant root primordia and from roots at different times after activation to the steady state of proliferation. The number, size and distribution of coiled bodies varied in the meristematic tissue depending on cell activity. While G0 cells in the dry primordia and proliferating cells showed a similar number of coiled bodies attached to their nucleoli, the number of nucleoplasmic coiled bodies greatly increased after the primordia were stimulated to proliferate. Their number remained steady from the time the meristematic population reached the steady state of proliferation, as estimated by flow cytometry. Fractionation studies demonstrated that coiled bodies are a part of the underlying nuclear matrix. Comparison of immunocytochemical and cytochemical data from confocal and electron microscopical studies demonstrated that the nucleolar and nucleoplasmic coiled bodies detected by confocal microscopy shared many features, suggesting that they form a family of closely related structures.     

小麦细胞核仁中DNA原位位置与rRNA基因转录位点的研究

以小麦细胞为研究材料, 应用常规电子显微镜技术和DNA细胞化学特异染色NAMA-Ur技术, 在原位水平对核仁中DNA的分布和特征进行了直观的观察。结果表明, 小麦细胞核仁中DNA位于纤维中心(Fibrillar Centers, FC)、致密纤维组分(Dense Fibrillar Component, DFC)以及两者的过渡区域, 并呈现出环绕FC排布的构型; 应用RNP优先染色(Benhard staining)技术分析了核仁中RNP的分布及其原位位置, 直观的显示了小麦细胞核仁中RNP颗粒主要集中在 FC与DFC的过渡区域及DFC和颗粒组分(Granular Component, GC)中; 并且在FC与DFC的过渡区域, 它们不太均匀也不太连续地半围绕着FC而排布; 进一步借助于RNA/DNA杂合体抗体在原位水平标记和分析了细胞核仁中活跃基因转录的精细位点, 结果表明小麦细胞核仁rRNA基因的转录位点位于FC与DFC的过渡区域及DFC中。     

Structure of the nucleoli of developing microsporangiate strobili and root tips of scotch pine

Summary The characteristics of the nucleoli of the microsporangiate strobili and the root tips of Scotch pine (Pinus sylvestris L.) vary both during the course of the cellular cycle and, with regard to the pattern and stage of organ and tissue differentiation. Nucleologenesis takes place in interphase and the nucleoli last until prophase. Several types of nucleoli occur during the nucleolar cycle, the pattern and age of tissues determining which type or types dominate. In the strobilus primordia collected at the end of July and in August, the mitotic frequency is high. Nucleoli remain small throughout the nucleolar cycle, and at the electron microscopic level, they display intermingled fibrillar and fibrillogranular components. Strobilus primordia collected in September contain larger nucleoli in the sporogenous nuclei than in the nuclei of the tapetum or of the wall cells. Amongst the nucleoli with completely intermingled fibrous and granular material, nucleoli with nucleolonema or with vacuoles occur frequently. Small balls of fibrous material are seen on the nucleolar surface and in the nucleoplasm. In October, the mitotic frequency of strobilal cells is low. Nucleoli with completely intermingled fibrillar and granular components have vanished whereas a new, compact type of nucleolus with a dense fibrillogranular main portion and with nucleolonema, has developed. The nucleoli of the sporogenous cells have enlarged continuously whereas those of the wall cells are small. The nucleoli of the root tip cell resemble, to a certain extent, those of the strobilus primordia collected in September. In squashed preparations, the nucleoli of the strobilal cells bind the common nucleolar stains poorly whereas the nucleoli of the root cells can be stained with all the methods used. In certain cases, DNase treatment improves the stainability of the strobilal nucleoli. AgNO₃-staining is successful after acetic acid: alcohol fixation but not after formalin: hydrochinone fixation.     

Cell cycle analysis and drug inhibition studies of silver staining in synchronous HeLa cells

Cytological staining with silver nitrate is specific for a protein associated with chromosomal nucleolus organizer regions and interphase nucleoli. At metaphase the amount of staining present is usually much less than that at interphase. During the transition from mitosis to G1, as seen in synchronized HeLa cells, the amount of silver staining increases and, by late G1, is located discretely and completely over the nucleolus. Such staining remains constant through G2. Towards late G2 a slight disorganization of the silver staining material is observed, possibly in preparation for the upcoming mitosis. Cells synchronized at mitosis and treated with either actinomycin D (AMD) or 2-mercapto-1-[2-(4-pyridyl)-ethyl]-benzimidazole (MPB), at concentrations which inhibit ribosomal RNA (rRNA) synthesis, show nucleolar fragmentation and little, if any, apparent increase in silver staining at early G1. After removal of the MPB, the nucleolar fragments reform nucleoli and the staining increases to control levels. Treatment of mitotic cells with puromycin dihydrochloride does not effect nucleolar morphology or the increase in silver staining. These results directly demonstrate that silver staining is associated with rRNA synthesis.     

In vivo dynamics and kinetics of pKi-67: transition from a mobile to an immobile form at the onset of anaphase

A cell proliferation marker protein, pKi-67, distributes to the chromosome periphery during mitosis and nucleolar heterochromatin in the interphase. We report here on the structural domains of pKi-67 that are required for its correct distribution. While both the LR domain and the conserved domain were involved in localization to the nucleolar heterochromatin, both the LR domain and the Ki-67 repeat domain were required for its distribution to the mitotic chromosome periphery. Using in vivo time-lapse microscopy, GFP-pKi-67 was dynamically tracked from the mitotic chromosome periphery to reforming nucleoli via prenucleolar bodies (PNBs). The signals in PNBs then moved towards and fused into the reforming nucleoli with a thin string-like fluorescence during early G1 phase. An analysis of the in vivo kinetics of pKi-67 using photobleaching indicated that the association of pKi-67 with chromatin was progressively altered from "loose" to "tight" after the onset of anaphase. These findings indicate that pKi-67 dynamically alters the nature of the interaction with chromatin structure during the cell cycle, which is closely related to the reformation process of the interphase nucleolar chromatin.     

REPOPULATION OF THE POSTMITOTIC NUCLEOLUS BY PREFORMED RNA

This study is concerned with the fate of the nucleolar contents, particularly nucleolar RNA, during mitosis Mitotic cells harvested from monolayer cultures of Chinese hamster embryonal cells, KB6 (human) cells, or L929 (mouse) cells were allowed to proceed into interphase in the presence or absence (control) of 0.04–0 08 µg/ml of actinomycin D, a concentration which preferentially inhibits nucleolar (ribosomal) RNA synthesis 3 hr after mitosis, control cells had large, irregularly shaped nucleoli which stained intensely for RNA with azure B and for protein with fast green. In cells which had returned to interphase in the presence of actinomycin D, nucleoli were segregated into two components easily resolvable in the light microscope, and one of these components stained intensely for RNA with azure B. Both nucleolar components stained for protein with fast green In parallel experiments, cultures were incubated with 0.04–0 08 µg/ml actinomycin D for 3 hr before harvesting of mitotic cells, then mitotic cells were washed and allowed to return to interphase in the absence of actinomycin D. 3 hr after mitosis, nuclei of such cells were devoid of large RNA-containing structures, though small, refractile nucleolus-like bodies were observed by phase-contrast microscopy or in material stained for total protein. These experiments indicate that nucleolar RNA made several hours before mitosis persists in the mitotic cell and repopulates nucleoli when they reform after mitosis     

In nucleoli, the steady state of nucleolar proteins is leptomycin B-sensitive.

BACKGROUND INFORMATION: The nucleolus is a dynamic structure. It has been demonstrated that nucleolar proteins rapidly associate with and dissociate from nucleolar components in continuous exchanges with the nucleoplasm using GFP (green fluorescent protein)-tagged proteins. However, how the exchanges within one nucleolus and between nucleoli within the nuclear volume occurred is still poorly understood. RESULTS: The movement of PAGFP (photoactivatable GFP)-tagged proteins that become visible after photoactivation can be followed. In the present study, we establish the protocol allowing quantification of the traffic of PAGFP-tagged nucleolar proteins in nuclei containing two nucleoli. The traffic in the activated area, at the periphery of the activated area and to the neighbouring nucleolus is measured. Protein B23 is rapidly replaced in the activated area, and at the periphery of the activated area the steady state suggests intranucleolar recycling of B23; this recycling is LMB (leptomycin B)-sensitive. The pool of activated B23 is equally distributed in the volume of the two nucleoli within 2 min. The three-dimensional distribution of the proteins Nop52 and fibrillarin is less rapid than that of B23 but is also LMB-sensitive. In contrast, traffic of fibrillarin from the nucleoli to the CB (Cajal body) was not modified by LMB. CONCLUSIONS: We propose that the steady state of nucleolar proteins in nucleoli depends on the affinity of the proteins for their partners and on intranucleolar recycling. This steady state can be impaired by LMB but not the uptake in the neighbouring nucleolus or the CB.     

REPOPULATION OF POSTMITOTIC NUCLEOLI BY PREFORMED RNA : II. Ultrastructure

The reconstruction of the nucleolus after mitosis was analyzed by electron microscopy in cultured mammalian (L929) cells in which nucleolar RNA synthesis was inhibited for a 3 h period either after or before mitosis. When synchronized mitotic cells were plated into a concentration of actinomycin D sufficient to block nucleolar RNA synthesis preferentially, nucleoli were formed at telophase as usual. 3 h after mitosis, these nucleoli had fibrillar and particulate components and possessed the segregated appearance characteristic of nucleoli of actinomycin D-treated cells. Cells in which actinomycin D was present for the last 3 h preceding mitosis did not form nucleoli by 3 h after mitosis though small fibrillar prenucleolar bodies were detectable at this time. These bodies subsequently grew in size and eventually acquired a particulate component. It took about a full cell cycle before nucleoli of these cells were completely normal in appearance. Thus, nucleolar RNA synthesis after mitosis is not necessary for organization of nucleoli after mitosis. However, inhibition of nucleolar RNA synthesis before mitosis renders the cell incapable of forming nucleoli immediately after mitosis. If cells are permitted to resume RNA synthesis after mitosis, they eventually regain nucleoli of normal morphology.     

In nucleoli, the steady state of nucleolar proteins is leptomycin B-sensitive

Background information. The nucleolus is a dynamic structure. It has been demonstrated that nucleolar proteins rapidly associate with and dissociate from nucleolar components in continuous exchanges with the nucleoplasm using GFP (green fluorescent protein)‐tagged proteins. However, how the exchanges within one nucleolus and between nucleoli within the nuclear volume occurred is still poorly understood. Results. The movement of PAGFP (photoactivatable GFP)‐tagged proteins that become visible after photoactivation can be followed. In the present study, we establish the protocol allowing quantification of the traffic of PAGFP‐tagged nucleolar proteins in nuclei containing two nucleoli. The traffic in the activated area, at the periphery of the activated area and to the neighbouring nucleolus is measured. Protein B23 is rapidly replaced in the activated area, and at the periphery of the activated area the steady state suggests intranucleolar recycling of B23; this recycling is LMB (leptomycin B)‐sensitive. The pool of activated B23 is equally distributed in the volume of the two nucleoli within 2 min. The three‐dimensional distribution of the proteins Nop52 and fibrillarin is less rapid than that of B23 but is also LMB‐sensitive. In contrast, traffic of fibrillarin from the nucleoli to the CB (Cajal body) was not modified by LMB. Conclusions. We propose that the steady state of nucleolar proteins in nucleoli depends on the affinity of the proteins for their partners and on intranucleolar recycling. This steady state can be impaired by LMB but not the uptake in the neighbouring nucleolus or the CB.     

Ultrastructural studies of the nucleoli in diploid and trisomic chickens

Chickens having a trisomic complement for the nucleolar organizer region (NOR) serve as a model for investigations on gene expression, molecular regulation, and physiological alterations resulting from extra genetic material. Of fundamental importance is the question of whether the extra set of ribosomal cistrons in trisomic chickens generates a morphologically and biochemically complete nucleolus. Ultrastructural and cytochemical studies of diploid and trisomic embryonic and adult cells were undertaken. All structures identified in trisomic cells as nucleoli by gross morphology were shown to be RNA rich by acridine orange fluorescence. At the ultrastructural level, the expected granular and fibrillar components were identified in all nucleoli of trisomic cells. Fibrillar centers, indicative of NOR's, were seen in all cases. These and other results from silver staining suggest full gene activity for each of the three chromosomes in the trisomic cell at all stages of development.     

THE NUCLEOLI IN MITOTIC DIVISIONS OF MAMMALIAN CELLS IN VITRO

In a number of mammalian cell strains nucleoli persisted through mitosis. This phenomenon was especially pronounced in several cell lines derived from Chinese hamster tissues. All the methods employed, including radioautography with tritiated uridine, cytochemical stains (methyl green-pyronin and azure B), fluorescent microscopy (coriphosphine O), ribonuclease digestion, and electron microscopy, demonstrated that the bodies identified as persistent nucleoli in the mitotic stages had the same characteristics as did the nucleoli in the interphase. Persistent nucleoli may attach to the chromosomes or may be free in the cytoplasm. In cells where no persistent nucleoli as such were noted, nucleolar material was observed to attach to the chromosomes in shapeless masses which moved with the chromosomes during anaphase. At least a portion of the nucleolar material was included in the daughter nuclei, presumably for immediate use for protein synthesis after cell division.     

The relationship of ribosomal RNA synthesis to the formation of segregated nucleoli and nucleolus-like bodies

The relationship of ribosomal RNA (rRNA) synthesis to nucleolar ultrastructure was studied in partial nucleolar mutants of Xenopus laevis. These mutations are the result of a partial deletion of rRNA genes and therefore alow studies on nucleolar structure and function without using drugs that inhibit rRNA synthesis. Ultrastructural studies demonstrated that normal embryos have reticulated nucleoli that are composed of a loose meshwork of granules and fibrils and a typical nucleolonema. In contrast, partial nucleolar mutants in which rRNA synthesis is reduced to less than 50% of the normal rate have compact nucleoli and nucleolus-like bodies. The compace nucleoli contain granules and fibrils, but they are segregated into distinct regions, and a nucleolonema is never seen. Since other species of RNA are synthesized normally by partial nucleolar mutants, these results demonstrate that nucleolar segragation is related specifically to a reduction in rRNA synthesis. The nucleolus-like bodies are composed mainly of fibrils,and the number of such bodies are composed mainly of fibrils, and the number of such bodies present in the different nucleolar mutants is inversely related to the relative rate of rRNA synthesis. Although the partial nucleolar organizers produce segregated nucleoli in these mutants, they organize morphologically normal, but smaller, nucleoli in heterozygous embryos. Alternative explanations to account for these results are discussed.     

Morphometry of nucleoli and expression of nucleolin analyzed by laser scanning cytometry in mitogenically stimulated lymphocytes.

BACKGROUND: Various attributes of nucleoli, including abundance of the nucleolar product (rRNA), correlate with cell-proliferative status and are useful markers for tumor diagnosis and prognosis. However, there is a paucity of methods that can quantitatively probe nucleolus. The aim of the present study was to utilize the morphometric capacity of the laser scanning cytometer (LSC) to analyze nucleoli and measure expression of the nucleolar protein nucleolin (NCL) in individual cells and correlate it with their state of proliferation. MATERIALS AND METHODS: Human lymphocytes were mitogenically stimulated, and at different time points their nucleoli were detected immunocytochemically using NCL Ab. The frequency of nucleoli per nucleus, their area, and the level of expression of NCL, separately in the nuclear and nucleolar compartments, were estimated in relation to the G(0) to G(1) transition and the cell cycle progression. RESULTS: During the first 24 h of stimulation, when the cells underwent G(0) to G(1) transition, their RNA content was increased nearly 8-fold, the level of NCL per nucleus also increased 8-fold, the NCL per nucleolus increased 12-fold, nucleolear area increased 3-fold, and NCL/nucleolar area increased nearly 4-fold. During the subsequent 24-48 h of stimulation, when cells were progressing through S, G(2), and M and reentering the next cycle, the number of nucleoli per nucleus was increased and a massive translocation of NCL from nucleoli to nucleoplasm was observed; its overall level per nucleus, however, still remained high, at 6-fold above of that of G(0) cells. CONCLUSIONS: While high expression of NCL in the nucleolar compartment correlates with the rate of rRNA accumulation in the cell and is a sensitive marker of the G(0) to G(1) transition, the cells progressing through the remainder of the cycle are better distinguished from G(0) cells by high overall level of NCL within the nucleus. Such an analysis, when applied to tumors, may be helpful in obtaining the quantitative parameters related to the kinetic status of the tumor-cell population and tumor prognosis. The capability of LSC to measure the protein translocation between nucleolus and nucleoplasm can be used to study the function and regulatory mechanisms of other proteins that reside in these compartments.     

Inhibition of nucleolar reformation after microinjection of antibodies to RNA polymerase I into mitotic cells

The formation of daughter nuclei and the reformation of nucleolar structures was studied after microinjection of antibodies to RNA polymerase I into dividing cultured cells (PtK2). The fate of several nucleolar proteins representing the three main structural subcomponents of the nucleolus was examined by immunofluorescence and electron microscopy. The results show that the RNA polymerase I antibodies do not interfere with normal mitotic progression or the early steps of nucleologenesis, i.e., the aggregation of nucleolar material into prenucleolar bodies. However, they inhibit the telophasic coalescence of the prenucleolar bodies into the chromosomal nucleolar organizer regions, thus preventing the formation of new nucleoli. These prenucleolar bodies show a fibrillar organization that also compositionally resembles the dense fibrillar component of interphase nucleoli. We conclude that during normal nucleologenesis the dense fibrillar component forms from preformed entities around nucleolar organizer regions, and that this association seems to be dependent on the presence of an active form of RNA polymerase I.     

Relations between nucleolar morphometric parameters and pre-rRNA synthesis in animal and plant cells

In order to study if there are differences between cells of the same tissue with one and two nucleoli, nuclear and nucleolar volume, density of tritiated uridine incorporation, amount of DNA per nucleus and intensity of cytoplasmic basophilia were measured in mononucleolated and binucleolated rat epithelial endometrial cells, in onion root meristematic cells and in chick embryo matrix cells of the central nervous system, neuroblasts and neurons. No significant differences in nuclear volume, density of tritiated uridine incorporation and amount of DNA per nucleus were found between cells of the same type with diverse numbers of nucleoli. Binucleolated endometrial cells, matrix cells, and root meristematic cells have biphasic distributions of nucleolar volumes. One peak of this distribution roughly coincides with the nucleolar volume of mononucleolated cells, the other peak corresponds almost to double the volume. As the density of uridine incorporation is the same irrespective of the nucleolar number and volume, the cells with larger nucleolar volumes have higher pre-rRNA synthesis. These cells also have higher amounts of ribosomes in the cytoplasm, as revealed by the photometric study of basophilia. It is concluded that in this population of cells the ribosomal production is regulated to a higher steady equilibrium than in the general population. This difference is not due to polyploidism or to the increased DNA content of G2 phase cells. Binucleolated neuroblasts and neurons have nucleolar volumes similar to those of mononucleolated ones.