Changes to cellular water and element content induced by nucleolar stress: investigation by a cryo-correlative nano-imaging approach

The cell is a crowded volume, with estimated mean mass percentage of macromolecules and of water ranging from 7.5 to 45 and 55 to 92.5 %, respectively. However, the concentrations of macromolecules and water at the nanoscale within the various cell compartments are unknown. We recently developed a new approach, correlative cryo-analytical scanning transmission electron microscopy, for mapping the quantity of water within compartments previously shown to display GFP-tagged protein fluorescence on the same ultrathin cryosection. Using energy-dispersive X-ray spectrometry (EDXS), we then identified various elements (C, N, O, P, S, K, Cl, Mg) in these compartments and quantified them in mmol/l. Here, we used this new approach to quantify water and elements in the cytosol, mitochondria, condensed chromatin, nucleoplasm, and nucleolar components of control and stressed cancerous cells. The water content of the control cells was between 60 and 83 % (in the mitochondria and nucleolar fibrillar centers, respectively). Potassium was present at concentrations of 128–462 mmol/l in nucleolar fibrillar centers and condensed chromatin, respectively. The induction of nucleolar stress by treatment with a low dose of actinomycin-D to inhibit rRNA synthesis resulted in both an increase in water content and a decrease in the elements content in all cell compartments. We generated a nanoscale map of water and elements within the cell compartments, providing insight into their changes induced by nucleolar stress.     

Ultrastructural aspects of nucleolar fibrillar centres in meristematic cells ofAllium cepa

Summary This paper deals with the fine structure of the fibrillar centres of the nucleolus inAllium cepa cells in ultrathin, sections of in vivo fixed roots. The ultrastructural observations have allowed us to consider each nucleolar fibrillar centre as an active zone in the nucleolar chromatin loop, and to propose a possible model for the organization of the different components of the nucleolus within it.Acknowledgments. The author wishes to thank Dr.M. C. Risueño for her valuable comments and discussion. This work has been partially supported by the III Plan de Desarrollo of Spain and by a grant of the Foundation Rodriguez Pascual.     

Observations on the dynamics of argyrophilic nucleolar material in the nuclei of mice spermatids

Applying a new silver staining technique it could be shown that in very early spermatids strong argyrophilia in nucleoli is confined to their granular and fibrillar components; fibrillar centers are devoid of silver. During subsequent developmental stages remnants of these nucleolar components are present in the form of intensively silver stained clusters of coiled fibers. As chromatin condensation proceeds, these fibrous structures decrease in size and density and are finally completely absent. The nucleus of the mature sperm contains only the space in which they formerly existed, now silver negative, as the so-called 'nuclear vacuole'.     

Observations on the dynamics of argyrophilic nucleolar material in the nuclei of mice spermatids

Summary Applying a new silver staining technique it could be shown that in very early spermatids strong argyrophilia in nucleoli is confined to their granular and fibrillar components; fibrillar centers are devoid of silver. During subsequent developmental stages remnants of these nucleolar components are present in the form of intensively silver stained clusters of coiled fibers. As chromatin condensation proceeds, these fibrous structures decrease in size and density and are finally completely absent. The nucleus of the mature sperm contains only the space in which they formerly existed, now silver negative, as the so-called nuclear vacuole.Acknowledgment. This investigation was supported partly by grant from the Universitätspreis der Wiener Wirtschaft.     

Nucleolar structure during the prophase of Physarum polycephalum]

With Sugihara's fixation technique, electron microscopic study of early-prophase nucleolus in the Physarm polycephalum showed the existence of particular fibrillar structures. The characteristic feature of these spherical structures is an electron-lucid center surrounded by a dense fibrillar component. Their relationships between the "fibrillar centers" and between the rate of ribosomal RNA synthesis are studied.     

Ultrastructural identification of the nucleolus organizer by the silver staining technic]

The nucleolus organizer regions can be selectively stained in metaphase chromosome preparations by the Goodpasture and Bloom's technique which was adapted to electron microscopy analysis of cells during interphase. Using this technique, a selective accumulation of silver grains was observed over nucleolus light areas. This selective accumulation allows the identification of the interphase fibrillar centers as the nucleolus organizer regions. Ultrastructural relationships between fibrillar centers and dense fibrillar component are discussed.     

Nucleolar control of p53: a cellular Achilles’ heel and a target for cancer therapy

Nucleoli perform a crucial cell function, ribosome biogenesis, and of critical relevance to the subject of this review, they are also extremely sensitive to cellular stresses, which can cause loss of function and/or associated structural disruption. In recent years, we have learned that cells take advantage of this stress sensitivity of nucleoli, using them as stress sensors. One major protein regulated by this role of nucleoli is the tumor suppressor p53, which is activated in response to diverse cellular injuries in order to exert its onco-protective effects. Here we discuss a model of nucleolar regulation of p53, which proposes that key steps in the promotion of p53 degradation by the ubiquitin ligase MDM2 occur in nucleoli, thus providing an explanation for the observed link between nucleolar disruption and p53 stability. We review current evidence for this compartmentalization in p53 homeostasis and highlight current limitations of the model. Interestingly, a number of current chemotherapeutic agents capable of inducing a p53 response are likely to do so by targeting nucleolar functions and these compounds may serve to inform further improved therapeutic targeting of nucleoli.     

Formaldehyde-Schiff's reagent as a nucleolar stain

Summary The use of formaldehyde-Schiff's reagent as a nucleolar stain has been described. Using different digestion procedures, it was confirmed that the stain is specific for RNA. It can be suitably used as a nucleolar stain, particularly in plant materials after a short TCA extraction, which probably extracts the nonbound RNA.     

Formaldehyde-Schiff's reagent as a nucleolar strain.

The use of formaldehyde-Schiff's reagent as a nucleolar stain has been described. Using different digestion procedures, it was confirmed that the stain is specific for RNA. It can be suitably used as a nucleolar stain, particularly in plant materials after a short TCA extraction, which probably extracts the nonbound RNA.     

Position of the nucleolus within the nuclei of pachytene spermatocytes of Dromiciops australis and Marmosa elegans (Didelphoidea-Marsupialia).

The location of the nucleoli within the nuclei of pachytene spermatocytes, and their relation with the position of the nucleolar organizer region (NOR) was studied. It appears that a terminal NOR determines a peripheral location of the nucleolus, due to the position of the NOR over the synaptonemal complex and to the attachment of the nucleolar chromosome telomeres at the nuclear membrane.     

Nucleolar localization and circadian regulation of Per2S,a novel splicing variant of the Period 2 gene

In this work, we show for the first time that a second splicing variant of the core clock gene Period 2 (Per2), Per2S, is expressed at both the mRNA and protein levels in human keratinocytes and that it localizes in the nucleoli. Moreover, we show that a reversible perturbation of the nucleolar structure acts as a resetting stimulus for the cellular clock. Per2S expression and periodic oscillation upon dexamethasone treatment were assessed by qRT-PCR using specific primers. Western blot (WB) analysis using an antibody against the recombinant human PER2 (abRc) displayed an intense band at a molecular weight of ~55 kDa, close to the predicted size of Per2S, and a weaker band at the expected size of Per2 (~140 kDa). The antibody raised against PER2 pS662 (abS662), an epitope absent in PER2S, detected only the higher band. Immunolocalization studies with abRc revealed a peculiar nucleolar signal colocalizing with the nucleolar marker nucleophosmin, whereas with abS662 the signal was predominantly diffuse all over the nucleus and partially colocalized with abRc in the nucleolus. The analysis of cell fractions by WB confirmed the enrichment of PER2S and the presence of PER2 in the nucleolar compartment. Finally, a pulse (1 h) of actinomycin D (0.01 μg/ml) induced reversible nucleolar disruption, PER2S de-localization and circadian synchronization of clock and Per2S genes. Our work represents the first evidence that the Per2S splicing isoform is a clock component expressed in human cells localizing in the nucleolus. These results suggest a critical role for the nucleolus in the process of circadian synchronization in human keratinocytes.     

Über die Ausschleusung nukleolärer Substanz im Milchdrüsenepithel der weissen Maus

Summary Mammary glands of the white mouse in different stages of lactation were fixed in Carnoy's fluid and the sections stained with Gallocyanin-chromalum für microscopic observation. After dislocation of the nucleolus to the nuclear membrane or the formation of a heterochromatic pathway between nucleolus and nuclear membrane the extrusion of nucleolar substance into the cytoplasm was observed.     

Detection of nucleolus organizer regions (NOR) in the chromosomes of the domestic pig (Sus scrofa domestica L.)

In the domestic pig (Sus scrofa domestica L.) nucleolar organizer regions (NOR) were detected by a combined silver-Giemsa method (Ag-G). The main sites of NORs are the secondary constrictions of chromosomes 8 and 10. Sometimes an additional NOR was observed near the centromere of 1 homologue of chromosome 11. Association of NORs were seen only between chromosomes 10 and at a very low frequency.     

Detection of nucleolus organizer regions (NOR) in the chromosomes of the domestic pig (Sus scrofa domestica L.)

Summary In the domestic pig (Sus scrofa domestica L.) nucleolar organizer regions (NOR) were detected by a combined silver-Giemsa method (Ag-G). The main sites of NORs are the secondary constrictions of chromosomes 8 and 10. Sometimes an additional NOR was observed near the centromere of 1 homologue of chromosome 11. Associations of NORs were seen only between chromosomes 10 and at a very low frequency.     

The nucleolar chromosome in embryos of Rana pipiens

Summary Karyotype analyses of prometaphases from medullary plate cells derived from mid-neurulae of Rana pipiens have led to the identification of the nucleolar chromosome and nucleolar organizing region, which is located on the longer arm of a small sub-metacentric chromosome (No. 10).Supported in part by research grant GB19631X from the Natioal Science Foundation. This study constituted a portion of a dissertation submitted by G. R. N. to the Medical College of Pennsylvania in partial fulfillment of requirements for a Ph. D. degree.Supported by NDEA and subsequently NSF predoctoral traineeships.To whom reprint requests should be sent.     

Létalité et anomalies du développement embryonnaire dans la descendance des femelles triploïdes de l'Amphibien UrodèlePleurodeles waltlii Michah

Summary A comparison is made between aneuploid off-springs of triploid females mated with diploid or triploid males ofPleurodeles waltlii. In the latter case, embryonic lethality is raised, and abnormalities are more severe. The disturbances affect morphogenesis, embryonic growth and physiological differentiation. At the cellular level, nuclear and nucleolar degenerative changes are conspicuous.     

Cytological evidence on the ability of the nucleolus organizing regions to assemble pre-existing nucleolar material

The treatment of Vicia faba root tips with 5-azacytidine caused unequal allotment of the chromosomes; after one cell cycle, a pair of daughter nuclei was produced, one of which carried the nucleolus organizing regions (NORs) and the other of which lacked them. The pre-existing nucleolar material brought into the NOR-carrying daughter nucleus by the chromosomes disappeared concurrently with the appearance of the new nucleoli, whereas in another daughter nucleus without the NOR a number of small nucleolus-like bodies became visible at random positions. These findings suggest that in addition to ribosomal RNA synthesis the NOR has another role in cells; to stimulate the pre-existing nucleolar material to assemble at the NOR itself.     

Cytological evidence on the ability of the nucleolus organizing regions to assemble pre-existing nucleolar material

Summary The treatment ofVicia faba root tips with 5-azacytidine caused unequal allotment of the chromosomes; after one cell cycle, a pair of daughter nuclei was produced, one of which carried the nucleolus organizing regions (NORs) and the other of which lacked them. The pre-existing nucleolar material brought into the NOR-carrying daughter nucleus by the chromosomes disappeared concurrently with the appearance of the new nucleoli, whereas in another daughter nucleus without the NOR a number of small nucleolus-like bodies became visible at random positions. These findings suggest that in addition to ribosomal RNA synthesis the NOR has another role in cells; to stimulate the pre-existing nucleolar material to assemble at the NOR itself.     

Comportamento delle fosfatasi alcaline all'azione di alcuni veleni carioclasici

Summary The effect of an aqueous solution (6 · 10^–3 M) of mustard oil on phosphatases was tested in cymotoid oocytes by treatment either of fixed and sectioned ovaries or of fresh gonads. Nuclear and cytoplasmic mono- and diphosphatases are destroyed; nucleolar phosphatases on the contrary are unaltered or only slightly damaged.The authors put forward the hypothesis that nucleolar phosphatases have a different kind of enzymatic active groups which might have a low competition factor in regard to mustard oil. Possibly the caryoclasic action of this substance is due, at least in part, to the destruction of alkaline phosphatases which are necessary to mitotic processes.