Localization of chromatin in "persistent" nucleoli in okadaic acid treated HeLa cells.

In okadaic acid treated HeLa cells, the chromosomes sometimes condense without being accompanied by nuclear envelope breakdown. These cells show "persistent" nucleoli. Within these "persistent" nucleoli the intranucleolar chromatin condenses and can be observed in the region of the dense nucleolar component (DNC) of the nucleoli. Other nucleolar components, namely the fibrillar centre (FC) and the granular component (GC) remain unchanged. These observations strongly speak for the localization of nucleolar chromatin (ribosomal cistrons) within the dense nucleolar component of the interphase nucleolus.     

细胞周期决定核糖体蛋白S6在真核细胞核仁中的聚积

核糖体蛋白S6（rpS6）是核糖体40S小亚基的核心组成蛋白之一。研究表明,rpS6可以通过核定位信号进入细胞核中,在核仁中参与核糖体的组装。在该研究中发现,rpS6在高等真核细胞核仁中的聚积与细胞周期有关,rpS6在S期中晚期开始在核仁中聚积,G2期含量达到最高,M期核仁分解时消失。推测,rpS6在核仁中的这种分布特性可能与核糖体的合成随细胞周期变化有关。     

Nucleolus: the fascinating nuclear body

Nucleoli are the prominent contrasted structures of the cell nucleus. In the nucleolus, ribosomal RNAs are synthesized, processed and assembled with ribosomal proteins. RNA polymerase I synthesizes the ribosomal RNAs and this activity is cell cycle regulated. The nucleolus reveals the functional organization of the nucleus in which the compartmentation of the different steps of ribosome biogenesis is observed whereas the nucleolar machineries are in permanent exchange with the nucleoplasm and other nuclear bodies. After mitosis, nucleolar assembly is a time and space regulated process controlled by the cell cycle. In addition, by generating a large volume in the nucleus with apparently no RNA polymerase II activity, the nucleolus creates a domain of retention/sequestration of molecules normally active outside the nucleolus. Viruses interact with the nucleolus and recruit nucleolar proteins to facilitate virus replication. The nucleolus is also a sensor of stress due to the redistribution of the ribosomal proteins in the nucleoplasm by nucleolus disruption. The nucleolus plays several crucial functions in the nucleus: in addition to its function as ribosome factory of the cells it is a multifunctional nuclear domain, and nucleolar activity is linked with several pathologies. Perspectives on the evolution of this research area are proposed.     

Structure and function of the nucleolus.

The activity of the ribosomal RNA genes generates a distinct subnuclear structure, the nucleolus, which is the site of ribosome biogenesis. The signals that target proteins and snoRNAs (small nucleolar RNAs) to the nucleolus, the nuclear import of ribosomal proteins, the export of the completed ribosomal subunits and the molecular organization of the nucleolus have been the subject of intense research during the past year. Evidence is accumulating that nucleoli functionally interact with coiled bodies and are also involved in the maturation of non-ribosomal RNA species.     

Diffusion-based transport of nascent ribosomes in the nucleus

Although the complex process of ribosome assembly in the nucleolus is beginning to be understood, little is known about how the ribosomal subunits move from the nucleolus to the nuclear membrane for transport to the cytoplasm. We show here that large ribosomal subunits move out from the nucleolus and into the nucleoplasm in all directions, with no evidence of concentrated movement along directed paths. Mobility was slowed compared with that expected in aqueous solution in a manner consistent with anomalous diffusion. Once nucleoplasmic, the subunits moved in the same random manner and also sometimes visited another nucleolus before leaving the nucleus.     

Partial inactivation of wheat nucleolus organisers by the nucleolus organiser chromosomes from Aegilops umbellulata

Two pairs of chromosomes (1U and 5U) in Aegilops umbellulata possess ribosomal RNA genes. This has been proven by studying wheat plants into which 1U and 5U chromosomes have been introduced separately. These plants have more ribosomal RNA genes than the recipient wheat plants and additional clusters of rDNA when examined by in situ hybridisation. The repeating rDNA unit in Aegilops umbellulata is longer than most of the units in the wheat variety Chinese Spring, the additional DNA probably being in the non-transcribed spacer. This was determined from restriction endonuclease maps of rDNA. In Chinese Spring plants possessing 1U or 5U chromosomes, the largest nucleoli are formed on 1U or 5U chromosomes and the wheat nucleolus organisers form micronucleoli. This is not because the nucleolus organisers on chromosomes 1U and 5U have many more rRNA genes than wheat nucleolus organisers. It is suggested that the Aegilops umbellulata nucleolus organisers are dominant over those of wheat because they compete more effectively for some limiting factor. — The partial inactivation of the wheat nucleolus organisers by chromosomes 1U or 5U does not result in a reduced total nucleolus volume in root tip or pollen mother cells, because of the compensation by the nucleolus organisers of chromosomes 1U or 5U. The amount of RNA in seedlings is not markedly affected by the partial inactivation of the wheat nucleolus organisers.     

Relative stoichiometry in ribosomal proteins in HeLa cell nucleoli.

Total protein was released from isolated HeLa cell nucleoli by guanidine hydrochloride, purified by cesium chloride density gradient centrifugation, and analyzed by two-dimensional polyacrylamide gel electrophoresis. Conditions of electrophoresis restricted attention to proteins that are positively charged at pH 8.6. Most of the major nucleolar protein spots co-electrophoresed with ribosomal proteins; the majority of ribosomal proteins from both the large and small ribosomal subunits were represented. Several proteins found in association with polysomes but not on ribosomal subunits and several proteins unique to the nucleolus were also identified in these nucleolar protein patterns. In order to determine whether the ribosomal proteins found in the nucleolus represented sizable pools of ribosomal proteins, or merely ribosomal proteins contained in the preribosomal particles, [35S]methionine-labeled nucleoli were mixed with [3H]methionine-labeled polysomes. From analysis of isotopic ratios in individual protein spots it was possible to determine the stoidchiometry of individual ribosomal proteins in the nucleolus relative to their complement on cytoplasmic ribosomes. All but a few proteins exhibited relative nucleolar stoichiometry values of approximately one, indicating that there are not significant pools of most ribosomal proteins in isolated nucleoli.     

核仁和核仁蛋白

核仁是位于细胞核内的非膜结构。电子显微镜下的核仁从形态上可以分为三层结构包括纤维中心区(FC)、高密度纤维区(DFC)和颗粒区(GC)。核仁内的蛋白有核糖体蛋白和非核糖体蛋白两种。利用蛋白质组学方法已经鉴定了350多种核仁蛋白,其中包括80多种核糖体蛋白。核仁是核糖体合成的场所,核仁中的非核糖体蛋白对核糖体的生物合成起关键调控作用。核仁不仅是细胞内通讯和核糖体：RNA加工的中心,而且在细胞周期、细胞增殖和衰老中起重要调控作用;核仁也是tRNA、mRNA和其它类型小分子RNA加工的场所。因此核仁是一个多功能的细胞生命活动中心。     

The nucleolus: an old factory with unexpected capabilities

The function of the nucleolus as a factory for assembling ribosomal subunits is well established, but many unrelated activities have been discovered over the past decade. Our understanding of the dynamics of nucleolar structure and its reassembly at the end of mitosis has recently advanced and the small nucleolar RNAs have been shown to be major players in the processing and modification of preribosomal RNA. Unexpectedly, the nucleolus also seems to play a role in nuclear export, sequestering regulatory molecules, modifying small RNAs, assembling ribonucleoprotein (RNP) and controlling aging.     

Distribution of newly formed ribosomal proteins in HeLa cell fractions

The distribution of newly formed ribosomal proteins between cytoplasmic, nucleoplasmic, and nucleolar fractions of HeLa cells was determined. All but a few of the newly formed ribosomal proteins were concentrated 10- to 50-fold in the nucleolus and two- to fivefold in the nucleoplasm. Nevertheless, substantial amounts were found in the cytoplasm. Pretreatment of cells with actinomycin D to deplete the nucleolar pool of ribosomal precursor RNA had no effect on the concentration of newly formed ribosomal proteins in the nucleus, but did lead to an increased amount in the nucleoplasm at the expense of the nucleolus.     

An investigation of some problems concerning nucleolus organizers in salamanders

Observed differences in the sizes of lampbrush nucleolus organizers in Plethodon cinereus have been shown by in situ hybridization to reflect true molecular differences in the numbers of ribosomal cistrons located at these organizers. Likewise, from in situ hybridization experiments on lampbrush and spermatocyte chromosomes it has been shown that animals may be, and indeed usually are, heterozygous with respect to the numbers of ribosomal cistrons on each half of the nucleolus bivalent. Filter hybridizations carried out on 33 males from a New Jersey population and 20 males from a Connecticut population have shown a 7.5-fold range in the numbers of ribosomal cistrons per diploid cell in the New Jersey population, and a 2.5-fold range in the Connecticut population. In view of the general heterozygosity of nucleolus organizers in these animals, the actual range in nucleolus organizer sizes in the New Jersey population is estimated to be at least 15-fold.     

The effect of actinomycin D on RNA synthesis and nucleolar ultrastructure in the liver of rats treated with fluorouracil

Summary Rats were given cytidine-³H and 10 min later 50 mg fluorouracil. They were killed after 25 hours. Actinomycin D was given at various times before sacrifice. The collapse of the nucleolus and the segregation of its components, seen in rats sacrificed one hour after administration of actinomycin D only, was prevented by prior treatment with fluorouracil. In rats treated with fluorouracil and given actinomycin 12 or 20 hours prior to death, there was a more or less pronounced collapse of the nucleolus but no typical segregation of its components. Radioautographs of livers from untreated rats or rats given actinomycin only at the times mentioned, and killed 25 hours after administration of cytidine-³H, were labelled mainly over the cytoplasm. Radioautographs from rats, treated with fluorouracil only, or fluorouracil plus actinomycin, showed labelling over the nucleoli, but depressed labelling over the cytoplasm. Biochemical analysis of RNA labelling showed high ribosomal peaks in untreated rats and rats treated with actinomycin only. Rats treated with fluorouracil, or fluorouracil plus actinomycin showed no labelling of the 29S and 18S ribosomal peaks. The results indicate that fluorouracil blocks or delays the formation of ribosomal RNA and that the inhibition, at least in part, takes place in the nucleolus.This work was supported by grants from the Swedish Medical Research Council (Project K68-12X-623-04), the Swedish Cancer Society (Project 6831), the Medical Faculty of Uppsala and the Swedish Society for Medical Research.     

Localization of chromosomal DNA sequences homologous to ribosomal gene type I insertion DNA in Drosophila melanogaster

Summary Chromosomal sites which have DNA homology to the 1 kb (kilobase pair) BamHI restrictable fragment of the 5 kb type I insertion present in many ribosomal genes in Drosophila melanogaster, were identified by using in situ hybridization and autoradiography. XX and XY complements of polytene chromosomes showed the nucleolus and chromocenter to be heavily labeled. Of the light label over euchromatic regions, the 102C band of chromosome 4 labeled particularly intensely. In mitotic XX and XY complements, the NORs (nucleolus organizer regions) of both sex chromosomes labeled as did the centromeric heterochromatin of autosomes. Label also appeared less frequently over telomeric and euchromatic regions.     

植物核糖体应激响应机制研究进展

核仁是真核细胞中重要的核结构,核糖体发生最初在核仁中进行,该过程涉及一系列复杂的反应,需要许多核仁相关因子参与。核糖体生物发生出现异常通常引起核仁结构紊乱,并导致细胞周期阻滞、细胞衰老甚至凋亡。核糖体应激响应机制在哺乳动物细胞中研究得较为深入,但在植物细胞中尚不明晰。尽管如此,人们逐渐发现某些植物特有的NAC转录因子家族成员在植物细胞中可能参与包括核糖体应激在内的多种胞内应激响应过程。此外,前期研究发现生长素系统与核糖体生物合成之间存在一种相互协调机制来调控植物发育。该文结合哺乳动物细胞中已知的核糖体应激响应通路,探讨植物细胞潜在的核糖体应激机制。     

Role of the nucleolus in plant cell response to environmental physical factors]

Contemporary data on ultrastructural reconstruction and changes in functional nucleolus activity in plant cells affected by physical environmental factors are presented. The main attention is paid to modifications of ribosomal gene expression under these conditions and induced changes in r-chromatin structure and ribosomal DNA localization. Recent data on fine nucleolus structure and molecular aspects of its organization as well as influence of microgravitation and clinostating on structural and functional organization of plant nucleoli are reported.     

Imaging of Nucleolar Dynamics During the Cell Cycle of Cancer Cells in Live Mice

The synthesis and assembly of ribosomal subunits take place in the nucleolus. The nucleolus forms in the nucleus around the repeated ribosomal gene clusters and undergoes cyclic changes during the cell cycle. Although the nucleolus is easily visualized by light microscopy of cells in vitro, the nucleolus has not been imaged in cells in vivo. We report here development of a mouse model to visualize the nucleolus cycle of cancer cells in live mice. HT-1080 human fibrosarcoma cells were labeled in the nucleus with histone H2B-GFP and with retroviral RFP in the cytoplasm. The nucleolus was visualized by contrast to the fluorescence of GFP expressed in the nucleus. HT-1080 dual-color cells were seeded on the surface of a skin-flap of nude mice. The inside surface of the skin-flap was directly imaged with a laser scanning microscope 24 hours after seeding. The nucleoli of the cancer cells were clearly imaged in real-time. The appearance of the nucleoli changed dramatically during the cell cycle. During mitosis, the nucleolus disappeared. After mitosis, the nucleoli decreased in number and increased in size. The nucleolus appears to have a major role in cell cycle regulation effected at least in part by sequestering proteins which affect cell cycle progression. Nucleolar imaging could be used for more precise determination of cancer-cell position in the cell cycle in vivo.