The Dynamic Organization of the Perinucleolar Compartment in the Cell Nucleus

The perinucleolar compartment (PNC) is a unique nuclear structure preferentially localized at the periphery of the nucleolus. Several small RNAs transcribed by RNA polymerase III (e.g., the Y RNAs, MRP RNA, and RNase P H1 RNA) and the polypyrimidine tract binding protein (PTB; hnRNP I) have thus far been identified in the PNC (Ghetti, A., S. PinolRoma, W.M. Michael, C. Morandi, and G. Dreyfuss. 1992. Nucleic Acids Res. 20:3671–3678; Matera, A.G., M.R. Frey, K. Margelot, and S.L. Wolin. 1995. J. Cell Biol. 129:1181–1193; Lee, B., A.G. Matera, D.C. Ward, and J. Craft. 1996. Proc. Natl. Acad. Sci. USA. 93: 11471–11476). In this report, we have further characterized this structure in both fixed and living cells. Detection of the PNC in a large number of human cancer and normal cells showed that PNCs are much more prevalent in cancer cells. Analysis through the cell cycle using immunolabeling with a monoclonal antibody, SH54, specifically recognizing PTB, demonstrated that the PNC dissociates at the beginning of mitosis and reforms at late telophase in the daughter nuclei. To visualize the PNC in living cells, a fusion protein between PTB and green fluorescent protein (GFP) was generated. Time lapse studies revealed that the size and shape of the PNC is dynamic over time. In addition, electron microscopic examination in optimally fixed cells revealed that the PNC is composed of multiple strands, each measuring ~80–180 nm diam. Some of the strands are in direct contact with the surface of the nucleolus. Furthermore, analysis of the sequence requirement for targeting PTB to the PNC using a series of deletion mutants of the GFP–PTB fusion protein showed that at least three RRMs at either the COOH or NH₂ terminus are required for the fusion protein to be targeted to the PNC. This finding suggests that RNA binding may be necessary for PTB to be localized in the PNC.     

Polytene chromosomes from ovarian pseudonurse cells of the Drosophila melanogaster otu mutant

Certain mutant alleles of the otu locus in Drosophila melanogaster produce abnormal nurse cells in the ovaries. These cells are called pseudonurse cells (PNC), since they generate polytene chromosomes instead of endopolyploid ones and do not normally have an oocyte to nurse. The banding pattern of polytene chromosome 3 from the salivary glands (SG) and from PNCs of homozygous otu ¹ females was compared and a detailed photomap of PNC chromosomes with different degrees of polyteny is presented. The banding pattern was found to be strikingly similiar in the two tissues. The puffing pattern of the PNC chromosomes was also studied and the function of the PNC chromosomes is discussed. No constrictions or breaks were found in the PNC chromosomes which seems to indicate that these sites, which are known to be underreplicated in the SG chromosomes, are equally replicated along with the rest of the chromosomes in the PNC nuclei.     

Platelet and neutrophil responses to Gram positive pathogens in patients with bacteremic infection

Background

Many Gram-positive pathogens aggregate and activate platelets in vitro and this has been proposed to contribute to virulence. Platelets can also form complexes with neutrophils but little is however known about platelet and platelet-neutrophil responses in bacterial infection.

Methodology/Principal Findings

We added isolates of Gram-positive bacteria from 38 patients with a bacteremic infection to blood drawn from the same patient. Aggregometry and flow cytometry were used to assess platelet aggregation and to quantify activation of platelets, neutrophils, and platelet-neutrophils complexes (PNCs) induced by the bacteria. Fifteen healthy persons served as controls. Most isolates of Staphylococcus aureus, beta hemolytic streptococci, and Enterococcus faecalis induced aggregation of platelets from their respective hosts, whereas pneumococci failed to do so. S. aureus isolates induced platelet aggregation more rapidly in patients than in controls, whereas platelet activation by S. aureus was lower in patients than in controls. PNCs were more abundant in baseline samples from patients than in healthy controls and most bacterial isolates induced additional PNC formation and neutrophil activation.

Conclusion/Significance

We have demonstrated for the first time that bacteria isolated from patients with Gram-positive bacteremia can induce platelet activation and aggregation, PNC formation, and neutrophil activation in the same infected host. This underlines the significance of these interactions during infection, which could be a target for future therapies in sepsis.     

Alpha and beta heterochromatin in polytene chromosome 2 ofDrosophila melanogaster

The formation of alpha and beta heterochromatin in chromosomes of Drosophila melanogaster was studied in salivary glands (SGs) and pseudonurse cells (PNCs). In SGs of X0, XY, XYY, XX and XXY individuals the amounts of alpha heterochromatin were similar, suggesting that the Y chromosome does not substantially contribute to alpha heterochromatin formation. Pericentric heterochromatin developed a linear sequence of blocks in PNCs, showing morphology of both alpha and beta heterochromatin. In situ hybridization with Rsp sequences (H _o clone) revealed that the most proximal heterochromatic segment of the mitotic map (region h39) formed a polytenized block in PNCs. Dot analysis showed that the clone had a hybridization rate with PNC-DNA very close to that with DNA from mainly diploid head cells, whereas the homologous SG-DNA was dramatically underrepresented. A similar increase of DNA representation in PNC was found for AAGAC satellite DNA. The mitotic region h44 was found not to polytenize in the SG chromosome, whereas in PNC chromosome 2 this region was partly polytenized and presented as an array of several blocks of alpha and beta heterochromatin. The mapping of deficiencies with proximal breakpoints in the most distal heterochromatin segments h35 in arm 2L and h46 in 2R showed that the mitotic eu-heterochromatin transitions were located in SG chromosomes distally to the polytene 40E and 41C regions, respectively. Thus, the transition zones between mitotic hetero- and euchromatin are located in banded polytene euchromatin. A scheme for dynamic organization of pericentric heterochromatin in nuclei with polytene chromosomes is proposed. Received: 17 November 1995; in revised form: 10 April 1996 / Accepted: 18 September 1996     

Cytophotometric studies on cells from the ovaries of otu mutants of Drosophila melanogaster

Summary Amounts of chromosomal DNA were estimated for Feulgen-stained, ovarian cells from flies carrying certain mutant alleles of the otu (ovarian tumor) gene. Epithelial sheath cells and lumen cells were found to contain the diploid (2C) amount of DNA and therefore served as internal, cytophotometric standards. Mitotically active follicle cells over young tumors-from homozygous otu ¹ females contained either the 2C or 4C amounts of DNA; whereas, the tumor cell population contained 2C, 4C and 8C nuclei and many intermediate values. Egg chambers also occur in homozygous otu ⁷ females. Follicle cells above these oocytes undergo a maximum of four cycles of endomitotic DNA replication. The accompanying nurse cells (PNC) contain polytene chromosomes. These undergo a maximum of 12 endonuclear replication cycles. The PNCs show the expected levels of DNA for the first 6 cycles and the fraction failing to replicate during subsequent cycles may be as small as 10%. Lower than expected levels of DNA were detected in PNCs from an otu ¹/otu ³ ovary, reflecting roughly 20% underreplication. The latter PNCs may have been interrupted before DNA synthesis was concluded. No simple model of genomic underreplication accounts for the several different patterns of DNA behavior observed for various otu mutants.     

Proteomic Identification and Characterization of a Novel Peroxisomal Adenine Nucleotide Transporter Supplying ATP for Fatty Acid β-Oxidation in Soybean and Arabidopsis

We have identified the novel protein Glycine max PEROXISOMAL ADENINE NUCLEOTIDE CARRIER (Gm PNC1) by proteomic analyses of peroxisomal membrane proteins using a blue native/SDS-PAGE technique combined with peptide mass fingerprinting. Gm PNC1, and the Arabidopsis thaliana orthologs At PNC1 and At PNC2, were targeted to peroxisomes. Functional integration of Gm PNC1 and At PNC2 into the cytoplasmic membranes of intact Escherichia coli cells revealed ATP and ADP import activities. The amount of Gm PNC1 in cotyledons increased until 5 d after germination under constant darkness and then decreased very rapidly in response to illumination. We investigated the physiological functions of PNC1 in peroxisomal metabolism by analyzing a transgenic Arabidopsis plant in which At PNC1 and At PNC2 expression was suppressed using RNA interference. The pnc1/2i mutant required sucrose for germination and suppressed the degradation of storage lipids during postgerminative growth. These results suggest that PNC1 contributes to the transport of adenine nucleotides that are consumed by reactions that generate acyl-CoA for peroxisomal fatty acid β-oxidation during postgerminative growth.     

Cytophotometric studies on cells from the ovaries of otu mutants of Drosophila melanogaster

Amounts of chromosomal DNA were estimated for Feulgen-stained, ovarian cells from flies carrying certain mutant alleles of the otu (ovarian tumor) gene. Epithelial sheath cells and lumen cells were found to contain the diploid (2C) amount of DNA and therefore served as internal, cytophotometric standards. Mitotically active follicle cells over young tumors from homozygous otu1 females contained either the 2C or 4C amounts of DNA; whereas, the tumor cell population contained 2C, 4C and 8C nuclei and many intermediate values. Egg chambers also occur in homozygous otu7 females. Follicle cells above these oocytes undergo a maximum of four cycles of endomitotic DNA replication. The accompanying nurse cells (PNC) contain polytene chromosomes. These undergo a maximum of 12 endonuclear replication cycles. The PNCs show the expected levels of DNA for the first 6 cycles and the fraction failing to replicate during subsequent cycles may be as small as 10%. Lower than expected levels of DNA were detected in PNCs from an otu1/otu3 ovary, reflecting roughly 20% underreplication. The latter PNCs may have been interrupted before DNA synthesis was concluded. No simple model of genomic underreplication accounts for the several different patterns of DNA behavior observed for various otu mutants.     

Lifetime of actin-dependent protein nanoclusters

Protein nanoclusters (PNCs) are dynamic collections of a few proteins that spatially organize in nanometer-length clusters. PNCs are one of the principal forms of spatial organization of membrane proteins, and they have been shown or hypothesized to be important in various cellular processes, including cell signaling. PNCs show remarkable diversity in size, shape, and lifetime. In particular, the lifetime of PNCs can vary over a wide range of timescales. The diversity in size and shape can be explained by the interaction of the clustering proteins with the actin cytoskeleton or the lipid membrane, but very little is known about the processes that determine the lifetime of the nanoclusters. In this paper, using mathematical modeling of the cluster dynamics, we model the biophysical processes that determine the lifetime of actin-dependent PNCs. In particular, we investigated the role of actin aster fragmentation, which had been suggested to be a key determinant of the PNC lifetime, and we found that it is important only for a small class of PNCs. A simple extension of our model allowed us to investigate the kinetics of protein-ligand interaction near PNCs. We found an anomalous increase in the lifetime of ligands near PNCs, which agrees remarkably well with experimental data on RAS-RAF kinetics. In particular, analysis of the RAS-RAF data through our model provides falsifiable predictions and novel hypotheses that will not only shed light on the role of RAS-RAF kinetics in various cancers, but also will be useful in studying membrane protein clustering in general.     

Synthesis of a water-stable CsPbBr3 perovskite for selective detection of mercury ion in water

By using the method of low-temperature crystallization, CsPbBr₃ perovskite nanocrystals (PNCs) coated with trifluoroacetyl lysine (Tfa-Lys) and oleamine (Olam) were synthesized in aqueous solution. The structure of the CsPbBr₃ PNCs was characterized by many methods, such as ultraviolet (UV)-visible absorption spectrophotometer, fluorescence spectrophotometer, transmission electron microscopy (TEM), and X-ray diffraction (XRD) pattern. The fluorescence emission of the CsPbBr₃ PNCs is stable in water for about 1 day at room temperature. It was also found that the fluorescence of the PNCs could be obviously and selectively quenched after the addition of mercury ion (Hg²⁺), allowing a visual detection of Hg²⁺ by the naked eye under UV light illumination. The fluorescence quenching rate (I₀/I) has a good linear relationship with the addition of Hg²⁺ in the concentration range 0.075 to 1.5 mg/L, with a correlation coefficient (R²) of 0.997, and limit of detection of 0.046 mg/L. The fluorescence quenching mechanism of the PNCs was determined by the fluorescence lifetime and X-ray photoelectron spectroscopy (XPS) of the PNCs. Overall, the synthesis method for CsPbBr₃ PNCs is simple and rapid, and the as-prepared PNCs are stable in water that could be conveniently used for selective detection of Hg²⁺ in the water environment.     

Dynamic organization of the β-heterochromatin in the Drosophila melanogaster polytene X chromosome

Region 20 of the polytene X chromosome of Drosophila melanogaster was studied in salivary glands (SG) and pseudonurse cells (PNC) of otu mutants. In SG chromosomes the morphology of the region strongly depends on two modifiers of position effect variegation: temperature and amount of heterochromatin. It is banded in XYY males at 25°?C and β-heterochromatic in X0 males at 14°?C, i.e. it shows dynamic transitions. In PNC chromosomes region 20 is not heterochromatic, but demonstrates a clear banding pattern. Some molecular markers of mitotic heterochromatin were localized by means of in situ hybridization on PNC chromosomes: DNA of the gene su(f) in section 20C, the nucleolar organizer and 359-bp satellite in 20F. The 359-bp satellite, which has been considered to be specific for heterochromatin of the mitotic X chromosome, was found at two additional sites on chromosome 3L, proximally to 80C. The right arm of the X chromosome in SG chromosomes was localized in the inversion In(1LR)pn2b: the telomeric HeT-A DNA and AAGAG satellite from the right arm are polytenized, having been relocated from heterochromatin to euchromatin.     

Chromosomal organization of Drosophila tumours

In otu mutants of Drosophila melanogaster ovarian tumours develop because of the high mitotic activity of the mutant cystocytes; the latter are normally endopolyploid. In certain alleles of otu, however, a varying proportion of the mutant ovarian cystocytes undergo polyteny. Mutant cystocytes with polytene chromosomes are termed pseudonurse cells (PNC). Polytene chromosome morphology and banding patterns in PNC of otu ¹/otu³ flies were cytologically analysed. Extensive variability was noted in the quality of the banding pattern of the PNC chromosomes which ranged from highly condensed (condensed PNC chromosomes) to those with a banding pattern (banded PNC chromosomes) similar to that in larval salivary gland cells (SGC). Both the condensed and banded PNC chromosomes frequently enter into a diffuse state characterised by weakened synapsis of the polytene chromatids and alterations in their banding pattern (diffuse PNC chromosomes). Analysis of DNA synthesis patterns in the various morphological forms of PNC polytene chromosomes by ³H-thymidine autoradiography revealed a basic similarity to the pattern seen in polytene nuclei of larval SGC. Independently replicating sites, however, could be unambiguously identified only in banded PNC chromosomes. Comparison of late replicating sites in such PNC chromosomes with those of larval SGC showed a remarkable similarity in the two cell types. These results suggest a close correlation between the polytene chromosome banding pattern and its replicative organization.     

CDK13, a Kinase Involved in Pre-mRNA Splicing,Is a Component of the Perinucleolar Compartment

The perinucleolar compartment (PNC) is a subnuclear stucture forming predominantly in cancer cells; its prevalence positively correlates with metastatic capacity. Although several RNA-binding proteins have been characterized in PNC, the molecular function of this compartment remains unclear. Here we demonstrate that the cyclin–dependent kinase 13 (CDK13) is a newly identified constituent of PNC. CDK13 is a kinase involved in the regulation of gene expression and whose overexpression was found to alter pre-mRNA processing. In this study we show that CDK13 is enriched in PNC and co-localizes all along the cell cycle with the PNC component PTB. In contrast, neither the cyclins K and L, known to associate with CDK13, nor the potential kinase substrates accumulate in PNC. We further show that CDK13 overexpression increases PNC prevalence suggesting that CDK13 may be determinant for PNC formation. This result linked to the finding that CDK13 gene is amplified in different types of cancer indicate that this kinase can contribute to cancer development in human.