Changes in nucleus, nucleolus and cell size accompanying somatic embryogenesis of Theobroma cacao L. I. Relationship between DNA and total protein content and size of nucleus, nucleolus and cell

There was a linear relation between an increase in DNA content and size of nuclei, nucleoli and cells in callus and proembryos (Theobroma cacao L.). In callus the increase of DNA content was accompanied by proportional increase in nuclear size whereas in proembryos the increase in nuclear size did not match the increasing amount of DNA. The stimulation of embryogenesis by 10(-2) mg/l 2,4-D was associated with increase in nuclear and nucleolar size and with decrease in cell sizes. Inhibition of embryogenesis by 1.0 mg/l 2,4-D+10% coconut water did not change nuclear size, but increased cell size in relation to the control. The process of embryo formation was accompanied by changes in relationship between nuclear, nucleolar and cell size and the total (DNFB-stained) proteins content. In callus as well as in proembryo the increase in total protein content in nucleus was not equivalent to the increasing sizes of nuclei which leads to the decrease in nuclear protein concentration. Similar situation was observed for nucleoli. Differences were found in the concentration of cytoplasmic proteins between the callus and proembryo cells. The stimulation of embryogenesis by low concentration of 2,4-D resulted in decrease in concentration of total proteins in nuclei and nucleoli and the increase in cytoplasm.     

Nuclear and nucleolar targeting sequences of c-erb-A, c-myb, N-myc, p53, HSP70, and HIV tat proteins

Protein import into the cell nucleus requires specific binding of nuclear proteins to the nuclear pore complex. Based on amino acid sequence "motifs" of known nuclear targeting signals, we identified peptides within a number of nuclear proteins with likely nuclear targeting potential and tested their function by transfecting into cells fusion genes that produce the cytoplasmic "reporter" protein, pyruvate kinase (PK), joined to the test sequence. Sequences within c-myb (PLLKKIKQ), N-myc (PPQKKIKS), p53 (PQPKKKP), and c-erb-A (SKRVAKRKL) oncoproteins that direct PK hybrids into the nucleus were identified. A peptide (GRKKRRQRRRAP) of the human immunodeficiency virus (HIV) tat protein (Tat), which contains two short basic regions, targets fusion proteins to the nucleolus. The COOH-terminal basic Tat region (QRRRAP) does not target PK hybrid proteins into the nucleus, but mutation of two basic amino acids in this region decreases but does not abolish nucleolar accumulation mediated by the entire Tat nucleolar targeting sequence. Moreover, the c-Myc nuclear targeting sequence fused to the COOH-terminal basic Tat region (PAAKRVKLDQRRRAP) effectively localizes PK hybrids to the nucleus and nucleolus. A similar sequence (FKRKHKKDISQNKRAVRR) in the human heat-shock protein HSP70 also localizes PK to the nucleus and nucleolus.     

Biogenesis of the signal recognition particle (SRP) involves import of SRP proteins into the nucleolus, assembly with the SRP-RNA, and Xpo1p-mediated export

The signal recognition particle (SRP) targets nascent secretory proteins to the ER, but how and where the SRP assembles is largely unknown. Here we analyze the biogenesis of yeast SRP, which consists of an RNA molecule (scR1) and six proteins, by localizing all its components. Although scR1 is cytoplasmic in wild-type cells, nuclear localization was observed in cells lacking any one of the four SRP "core proteins" Srp14p, Srp21p, Srp68p, or Srp72p. Consistently, a major nucleolar pool was detected for these proteins. Sec65p, on the other hand, was found in both the nucleoplasm and the nucleolus, whereas Srp54p was predominantly cytoplasmic. Import of the core proteins into the nucleolus requires the ribosomal protein import receptors Pse1p and Kap123p/Yrb4p, which might, thus, constitute a nucleolar import pathway. Nuclear export of scR1 is mediated by the nuclear export signal receptor Xpo1p, is distinct from mRNA transport, and requires, as evidenced by the nucleolar accumulation of scR1 in a dis3/rrp44 exosome component mutant, an intact scR1 3' end. A subset of nucleoporins, including Nsp1p and Nup159p (Rat7p), are also necessary for efficient translocation of scR1 from the nucleus to the cytoplasm. We propose that assembly of the SRP requires import of all SRP core proteins into the nucleolus, where they assemble into a pre-SRP with scR1. This particle can then be targeted to the nuclear pores and is subsequently exported to the cytoplasm in an Xpo1p-dependent way.     

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.     

Nucleolar localization/retention signal is responsible for transient accumulation of histone H2B in the nucleolus through electrostatic interactions

The majority of known nuclear proteins are highly mobile. The molecular mechanisms by which they accumulate inside stable compartments that are not separated from the nucleoplasm by membranes are obscure. The compartmental retention of some proteins is associated with their biological function; however, some protein interactions within distinct nuclear structures may be non-specific. The non-specific retention may lead to the accumulation of proteins in distinct structural domains, even if the protein does not function inside this domain. In this study, we have shown that histone H2B-EGFP initially accumulated in the nucleolus after ectopic expression, and then gradually incorporated into the chromatin to leave only a small amount of nucleolus-bound histone that was revealed by removing chromatin-bound proteins with DNase I treatment. Nucleolar histone H2B had several characteristics: (i) it preferentially bound to granular component of the nucleolus and interacted with RNA or RNA-containing nucleolar components; (ii) it freely exchanged between the nucleolus and nucleoplasm; (iii) it associated with the nuclear matrix; and (iv) it bound to interphase prenuclear bodies that formed after hypotonic treatment. The region in histone H2B that acts as a nucleolar localization/retention signal (NoRS) was identified. This signal overlapped with a nuclear localization signal (NLS), which appears to be the primary function of this region. The NoRS activity of this region was non-specific, but the molecular mechanism was probably similar to the NoRSs of other nucleolar proteins. All known NoRSs are enriched with basic amino acids, and we demonstrated that positively charged motifs (nona-arginine (R9) and nona-lysine (K9)) were sufficient for the nucleolar accumulation of EGFP. Also, the correlation between measured NoRS activity and the predicted charge was observed. Thus, NoRSs appear to achieve their function through electrostatic interactions with the negatively charged components of the nucleolus. Though these interactions are non-specific, the functionally unrelated retention of a protein can increase the probability of its interaction with specific and functionally related binding sites.     

In vitro development of early proembryos and plant regeneration via microculture in Oryza sativa

Two convenient and efficient microculture techniques (liquid droplet and shallow-layered culture) were used to rear 2-day-old and 3 to 4 day-old proembryos in rice. Among four cultivars, growth rate and frequency of embryogenesis were higher in the japonica cultivars than in the indica cultivars during proembryo culture. Two-day-old proembryos could grow and form callus only in Km8p and N6 among four kinds of tested media, and plantlets regenerated via organogenesis. Plant regeneration from callus initiated from three- and four-day-old proembryos occurred through somatic embryogenesis and organogenesis. For in vitro embryogenesis it was essential to supplement the medium with 14 amino acids and coconut milk. The highest frequency of embryogenesis and the frequency of total induction after 14 days of culture were approximately 42% and 95% for 3-day-old proembryos, and 45% and 100% for 4-day-old proembryos, respectively. This revised version was published online in June 2006 with corrections to the Cover Date.     

Determination of the functional domains involved in nucleolar targeting of nucleolin.

Nucleolin (713 aa), a major nucleolar protein, presents two structural domains: a N-terminus implicated in interaction with chromatin and a C-terminus containing four RNA-binding domains (RRMs) and a glycine/arginine-rich domain mainly involved in pre-rRNA packaging. Furthermore, nucleolin was shown to shuttle between cytoplasm and nucleolus. To get an insight on the nature of nuclear and nucleolar localization signals, a set of nucleolin deletion mutants in fusion with the prokaryotic chloramphenicol acetyltransferase (CAT) were constructed, and the resulting chimeric proteins were recognized by anti-CAT antibodies. First, a nuclear location signal bipartite and composed of two short basic stretches separated by eleven residues was characterized. Deletion of either motifs renders the protein cytoplasmic. Second, by deleting one or more domains implicated in nucleolin association either with DNA, RNA, or proteins, we demonstrated that nucleolar accumulation requires, in addition to the nuclear localization sequence, at least two of the five RRMs in presence or absence of N-terminus. However, in presence of only one RRM the N-terminus allowed a partial targeting of the chimeric protein to the nucleolus.     

Cytodifferentiation of the chick pancreas. 3. The content and synthesis of ribonucleic acid and proteins in developing acinar cells

The content and synthesis of ribonucleic acid (RNA) and protein was studied by microphotometry and autoradiography in the developing pancreatic acinar cells of White Leghorn chick embryos. These findings were correlated with previously reported changes in ultrastructural components. Shortly before or concomitant with zymogen granulation, RNA synthesis increased, in association with increases in the amount of nucleolar and cytoplasmic protein. The cytoplasmic fraction was transitory, whereas the accumulated nucleolar protein was maintained and was soon followed by an increase in nucleolar RNA. Concomitantly, a decrease in chromosomal RNA was observed, with the total amount of nuclear RNA staying constant. When zymogen first appeared, nucleoli were greatly enlarged due to large amounts of RNA and protein; total cellular RNA and protein had decreased slightly, in association with a decrease in cell volume. Subsequent development presented smaller nucleoli with decreased amounts of RNA and protein. Total cellular RNA increased due to its accumulation in the cytoplasm, probably as ribosomes. The accumulation of zymogen and the enlargement of other cellular structures contributed to an increase in total cellular protein. Prior to hatching, total cell RNA and protein decreased in amount, probably due to a reduction in cell volume through cell division.     

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.     

Cell-cycle-dependent localization of human cytomegalovirus UL83 phosphoprotein in the nucleolus and modulation of viral gene expression in human embryo fibroblasts in vitro

The nucleolus is a multifunctional nuclear compartment widely known to be involved in several cellular processes, including mRNA maturation and shuttling to cytoplasmic sites, control of the cell cycle, cell proliferation, and apoptosis; thus, it is logical that many viruses, including herpesvirus, target the nucleolus in order to exploit at least one of the above-mentioned functions. Recent studies from our group demonstrated the early accumulation of the incoming ppUL83 (pp65), the major tegument protein of human cytomegalovirus (HCMV), in the nucleolus. The obtained results also suggested that a functional relationship might exist between the nucleolar localization of pp65, rRNA synthesis, and the development of the lytic program of viral gene expression. Here we present new data which support the hypothesis of a potentially relevant role of HCMV pp65 and its nucleolar localization for the control of the cell cycle by HCMV (arrest of cell proliferation in G1-G1/S), and for the promotion of viral infection. We demonstrated that, although the incoming pp65 amount in the infected cells appears to be constant irrespective of the cell-cycle phase, its nucleolar accumulation is prominent in G1 and G1/S, but very poor in S or G2/M. This correlates with the observation that only cells in G1 and G1/S support an efficient development of the HCMV lytic cycle. We propose that HCMV pp65 might be involved in regulatory/signaling pathways related to nucleolar functions, such as the cell-cycle control. Co-immunoprecipitation experiments have permitted to identify nucleolin as one of the nucleolar partners of pp65.     

Proteomic analysis of the Arabidopsis nucleolus suggests novel nucleolar functions

The eukaryotic nucleolus is involved in ribosome biogenesis and a wide range of other RNA metabolism and cellular functions. An important step in the functional analysis of the nucleolus is to determine the complement of proteins of this nuclear compartment. Here, we describe the first proteomic analysis of plant (Arabidopsis thaliana) nucleoli, in which we have identified 217 proteins. This allows a direct comparison of the proteomes of an important nuclear structure between two widely divergent species: human and Arabidopsis. The comparison identified many common proteins, plant-specific proteins, proteins of unknown function found in both proteomes, and proteins that were nucleolar in plants but nonnucleolar in human. Seventy-two proteins were expressed as GFP fusions and 87% showed nucleolar or nucleolar-associated localization. In a striking and unexpected finding, we have identified six components of the postsplicing exon-junction complex (EJC) involved in mRNA export and nonsense-mediated decay (NMD)/mRNA surveillance. This association was confirmed by GFP-fusion protein localization. These results raise the possibility that in plants, nucleoli may have additional functions in mRNA export or surveillance.     

Morphometry of nuclear and nucleolar structures in a CaCo-2 cell line

The number of the nucleoli in a CaCo-2 cell nucleus does not generally depend on the quantity of DNA in the nucleus, but nucleolar DNA content is directly proportional to total nuclear DNA. However, in multinucleolar cells (three or more nucleoli), the nucleolar DNA content increases after 96 h incubation in culture without concomitant quantitative changes in nuclear DNA. The percentage of multinucleolar cells and the average number of nucleoli per nucleus increase with increasing incubation time. After 72 and 96 h in culture, multinucleolar cells show distinctive morphologies. The ratio of the sum of nucleolar perimeters to the nuclear perimeter increases linearly when the number of nucleoli in a nucleus increases, but there is no concomitant increase in total nucleolar area or DNA content, except in the 72 and 96 h populations. When the number of nucleoli in CaCo-2 cells increases after 48 and 60 h in culture, the amount of DNA per nucleolus decreases.     

The cytological changes of tobacco zygote and proembryo cells induced by beta-glucosyl Yariv reagent suggest the involvement of arabinogalactan proteins in cell division and cell plate formation

ABSTRACT: BACKGROUND: In dicotyledonous plant, the first asymmetric zygotic division and subsequent several cell divisions are crucial for proembryo pattern formation and later embryo development.. Arabinogalactan proteins (AGPs) are a family of extensively glycosylated cell surface proteins that are thought to have important roles in various aspects of plant growth and development, including embryogenesis. Previous results from our laboratory show that AGPs are concerned with tobacco egg cell fertilization and zygotic division. However, how AGPs interact with other factors involved in zygotic division and proembryo development remains unknown. RESULTS: In this study, we used the tobacco in vitro zygote culture system and series of meticulous cell biology techniques to investigate the roles of AGPs in zygote and proembryo cell division. For the first time, we examined tobacco proembryo division patterns detailed to every cell division. The bright-field images and statistical results both revealed that with the addition of an exogenous AGPs inhibitor, beta-glucosyl Yariv (beta-GlcY) reagent, the frequency of aberrant division increased remarkably in cultured tobacco zygotes and proembryos, and the cell plate specific locations of AGPs were greatly reduced after beta-GlcY treatment. In addition, the accumulations of new cell wall materials were also significantly affected by treating with beta-GlcY. Detection of cellulose components by Calcofluor white stain showed that strong fluorescence was located in the newly formed wall of daughter cells after the zygotic division of in vivo samples and the control samples from in vitro culture without beta-GlcY treatment; while there was only weak fluorescence in the newly formed cell walls with beta-GlcY treatment. Immunocytochemistry examination with JIM5 and JIM7 respectively against the low- and high-esterified pectins displayed that these two pectins located in opposite positions of zygotes and proembryos in vivo and the polarity was not affected by beta-GlcY. Furthermore, FM4-64 staining revealed that endosomes were distributed in the cell plates of proembryos, and the localization pattern was also affected by beta-GlcY treatment. These results were further confirmed by subsequent observation with transmission electron microscopy. Moreover, the changes to proembryo cell-organelles induced by beta-GlcY reagent were also observed using fluorescent dye staining technique. CONCLUSIONS: These results imply that AGPs may not only relate to cell plate position decision, but also to the location of new cell wall components. Correlated with other factors, AGPs further influence the zygotic division and proembryo pattern establishment in tobacco.     

Control of ribosome biosynthesis in plant cell cultures under heat shock conditions. II. Ribosomal proteins

The nomenclature and synthesis of acidic and basic ribosomal proteins of plant cell cultures are described, with special regard to ribosome biosynthesis under control and heat-shock conditions. Assembly and processing of preribosomes in the nucleolus require a defined set of ribosomal proteins binding to the nascent pre-rRNA chain. Others are added later on the maturation pathway, mostly in the cytoplasm. Although, under appropriate heat-shock conditions, formation of mature ribosomes is completely blocked, most of the typical ribosomal proteins are still detected in the nuclear fraction. They are constituents of heat-shock preribosomes, which can be processed to normal cytoplasmic ribosomes only if the cells are allowed to recover at 25°C shortly after the labeling period at 40°C. However, if hyperthermic conditions are maintained, the labeled pre-rRNP material is evidently partly broken down. It forms the growing amount of RNP granules (ribosomal wastage) characteristic of the dispersed nucleolus of heat-shocked cells. In addition to the ‘nucleolar’ ribosomal proteins, a few newly formed ribosomal proteins can also be detected in cytoplasmic ribosomes under heat-shock conditions. Most of them belong to the group of exchange proteins whose labeling continues even if pre-rRNA synthesis is blocked by actinomycin D.