Characterization of endogenous human promyelocytic leukemia isoforms.

Promyelocytic leukemia (PML) has been implicated in a variety of functions, including control of TP53 function and modulation of cellular senescence. Sumolated PML is the organizer of mature PML bodies, recruiting a variety of proteins onto these nuclear domains. The PML gene is predicted to encode a variety of protein isoforms.

TRAF interactions with raft-like buoyant complexes, better than TRAF rates of degradation, differentiate signaling by CD40 and EBV latent membrane protein 1.

The CD40 receptor and the Epstein-Barr virus oncoprotein LMP1 are both members of the TNF-receptor family and share several signaling mediators, including TRAF2 and TRAF3. Depending on the cell lineage and stage of maturation, LMP1 and CD40 can have synergistic, antagonist or unrelated effects. Previous publications have suggested that both TRAF2 and TRAF3 move into lipid rafts upon LMP1 expression or CD40 activation, whereas their proteolysis is only enhanced by CD40.

Adenovirus protein IX sequesters host-cell promyelocytic leukaemia protein and contributes to efficient viral proliferation.

The product of adenovirus type 5 (Ad5) gene IX, protein IX (pIX), is a multifunctional protein that stabilizes the viral capsid and has transcriptional activity. We show that pIX also contributes to the Ad5-induced reorganization of the host-cell nuclear ultrastructure: pIX induces the formation of specific and dynamic nuclear inclusions, and the host promyelocytic leukaemia (PML) protein, which is the main structural organizer of PML bodies, is stably relocated and confined within the pIX-induced inclusions late in infection. Our results suggest that Ad5 has evolved a unique strategy that leads to the sustained neutralization of PML bodies throughout infection, thereby ensuring optimal viral proliferation.

The relationship between BCL6 bodies and nuclear sites of normal and halogenated DNA and RNA synthesis.

BCL6 is a POZ/BTB and zinc finger transcription factor that self-interacts and accumulates into discrete nuclear "bodies" of unknown function. We recently reported that BCL6 bodies associate with bromodeoxyuridine (BrdU)-substituted DNA, suggesting their implication in replication. To examine this possibility, we examine here by electron and confocal microscopy the relation between BCL6 bodies and replication foci (RF) using incorporation of various halogenated nucleotides (BrdU, chlorodeoxyuridine, CldU, and iododeoxyuridine, IdU) or PCNA (proliferating cell nuclear antigen) staining.

Class II histone deacetylases are directly recruited by BCL6 transcriptional repressor.

BCL6 is a member of the POZ/zinc finger (POK) family involved in survival and/or differentiation of a number of cell types and in B cell lymphoma upon chromosomal alteration. Transcriptional repression by BCL6 is thought to be achieved in part by recruiting a repressor complex containing two class I histone deacetylases (HDACs). In this study we investigated whether BCL6 could also target members of class II HDACs.

Adenovirus infection targets the cellular protein kinase CK2 and RNA-activated protein kinase (PKR) into viral inclusions of the cell nucleus.

The effects of the adenovirus infection on the distribution of the cellular protein kinase CK2 and double-stranded RNA-activated protein kinase (PKR) were examined at the ultrastructural level. Immunogold labeling revealed the redistribution of CK2 subunits and PKR to morphologically distinct structures of the cell nucleus. The electron-clear amorphous structures, designated pIX nuclear bodies in our previous work (Rosa-Calatrava et al.

Functional analysis of adenovirus protein IX identifies domains involved in capsid stability, transcriptional activity, and nuclear reorganization.

The product of adenovirus (Ad) type 5 gene IX (pIX) is known to actively participate in the stability of the viral icosahedron, acting as a capsid cement. We have previously demonstrated that pIX is also a transcriptional activator of several viral and cellular TATA-containing promoters, likely contributing to the transactivation of the Ad expression program. By extensive mutagenesis, we have now delineated the functional domains involved in each of the pIX properties: residues 22 to 26 of the highly conserved N-terminal domain are crucial for incorporation of the protein into the virion; specific residues of the C-terminal leucine repeat are responsible for pIX interactions with itself and possibly other proteins, a property that is critical for pIX transcriptional activity.

Isolation and characterization of an equine foamy virus.

Foamy viruses (FVs) are complex retroviruses which have been isolated from different animal species including nonhuman primates, cattle, and cats. Here, we report the isolation and characterization of a new FV isolated from blood samples of horses. Similar to other FVs, the equine foamy virus (EFV) exhibits a highly characteristic ultrastructure and induces syncytium formation and subsequent cell lysis on a large number of cell lines.

The cell-surface-expressed nucleolin is associated with the actin cytoskeleton.

Nucleolin is a RNA- and protein-binding multifunctional protein. Mainly characterized as a nucleolar protein, nucleolin is continuously expressed on the surface of different types of cells along with its intracellular pool within the nucleus and cytoplasm. By confocal and electron microscopy using specific antibodies against nucleolin, we show that cytoplasmic nucleolin is found in small vesicles that appear to translocate nucleolin to the cell surface.

DNA replication progresses on the periphery of nuclear aggregates formed by the BCL6 transcription factor.

The BCL6 proto-oncogene, frequently alterated in non-Hodgkin lymphoma, encodes a POZ/zinc finger protein that localizes into discrete nuclear subdomains. Upon prolonged BCL6 overexpression in cells bearing an inducible BCL6 allele (UTA-L cells), these subdomains apparently coincide with sites of DNA synthesis. Here, we explore the relationship between BCL6 and replication by both electron and confocal laser scanning microscopy.

Deletion of the fiber gene induces the storage of hexon and penton base proteins in PML/Sp100-containing inclusions during adenovirus infection.

The present study has documented changes in the in situ distribution of viral DNA and capsid proteins in 293 cells infected with fiber gene-deleted adenoviruses. It shows that infection results in the intense production of progeny viruses which appear morphologically intact although they are devoid of fiber-coding sequence in their genome and hence of fiber protein in their capsid. The data confirm, therefore, that fiber protein is not essential for the assembly of progeny viruses.

An anchorage nuclear structure for telomeric DNA repeats in HeLa cells.

Using either a biotinylated peptide nucleic acid (PNA) oligomer or a digoxigenin-labeled double-stranded DNA probe, we determined the distribution of the telomeric DNA repeats in HeLa cells by in-situ hybridization at the ultrastructural level. The telomeric DNA was found at the periphery of previously unrecognized roundish nuclear structures, distributed throughout the nucleoplasm. The levels of association of the telomeric DNA with these structures was investigated by exposure of cells to a detergent-containing hypotonic solution which only preserves tightly linked components.

Release of viruses and viral DNA from nucleus to cytoplasm of HeLa cells at late stages of productive adenovirus infection as revealed by electron microscope in situ hybridization.

Considerable progress has been made over the past 10 years towards a full understanding of the functional significance of the structural changes resulting from the production of adenoviruses in permissive cells. Similarly, the host-virus interactions which are involved in viral replication and gene expression as well as in RNA nuclear export have been investigated. Post-embedding nonisotopic in situ hybridization has been proven to be a powerful tool for the study of nucleic acids in infected cells provided that controlled elimination of artifacts by appropriate treatments was undertaken.

Pilot ultrastructural evaluation of human preauricular skin before and after high-energy pulsed carbon dioxide laser treatment.

Background: Carbon dioxide laser resurfacing has recently come into favor for the treatment of photodamaged skin. While the clinical and histologic effects of high-energy short-pulse carbon dioxide lasers on human skin have been investigated, the ultrastructural effects of these lasers have not been documented. Our objective was to study the ultrastructural effects of a high-energy pulsed carbon dioxide laser on photodamaged human skin.

Ultrastructural localization of interferon-inducible double-stranded RNA-activated enzymes in human cells.

The protein kinase PKR and the 2',5'-oligoadenylate (2-5A) synthetase are two interferon-induced and double-stranded RNA-activated enzymes which are implicated in the mechanism of action of interferon. Their distribution was undertaken here at the ultrastructural level by the immunogold procedure, following the use of specific monoclonal antibodies directed against PKR and 69- and 100-kDa forms of the 2-5A synthetase. These enzymes were detected as a pool of nonaggregated proteins scattered throughout the cell and as aggregates often associated with electron-dense doughnut-like structures showing a similar aspect whatever their subcellular localization: the cytoplasm, the nuclear envelope, and the nucleus.

Early stages of pre-rRNA formation within the nucleolar ultrastructure of mouse cells studied by in situ hybridization with a 5'ETS leader probe.

The first cleavage in the processing of the rRNA primary transcript in mammals occurs within the 5'-terminal region of the 5' external transcribed spacer (5'ETS), which makes the upstream portion of this spacer a selective marker of nascent transcripts. Moreover, short treatments with low doses of actinomycin D (AMD), which selectively suppress pre-rRNA synthesis and allow processing of preformed pre-rRNAs, result in the production of prematurely terminated transcripts essentially spanning the 5'ETS leader region. To gain further insight into the intranucleolar localization of early stages of preribosome formation we analyzed the distribution of this specific pre-rRNA segment by in situ hybridization at the ultrastructural level in AMD-treated or in control 3T3 mouse cells.

The ultrastructural localization of 60-kDa Ro protein and human cytoplasmic RNAs: association with novel electron-dense bodies.

The 60-kDa Ro ribonucleoprotein is an important target of humoral autoimmune responses. However, the ultrastructural locations of the 60-kDa Ro protein and its associated small cytoplasmic RNAs (Y RNAs) have not been previously determined, and the functions of the Ro protein and RNAs are not known. In this study, the cellular locations of the 60-kDa Ro protein and the Ro Y1 and Y4 RNAs are determined by immunoelectron microscopy and in situ hybridization electron microscopy, respectively.

Nuclear targeting of incoming human foamy virus Gag proteins involves a centriolar step.

The pathways used in the transport of retroviral genomes to the nucleus are poorly identified. Analyzing the intracellular localization of incoming foamy viruses, we have found that the Gag antigens and the viral genome accumulate in a distinct perinuclear domain identified as the centrosome. Colchicine treatment completely abolished pericentriolar targeting of human foamy virus (HFV) proteins, suggesting a role for microtubules in the transport of the incoming viral proteins to the centrioles.

Identification of transcription factories in nuclei of HeLa cells transiently expressing the Us11 gene of herpes simplex virus type 1.

Nuclear distribution and migration of herpes simplex virus type 1 Us11 transcripts were studied in transient expression at the ultrastructural level and compared to that of RNA polymerase II protein. Transcription was monitored by autoradiography following a short pulse with tritiated uridine. Us11 transcripts accumulated mainly over the foci of intermingled RNP fibrils as demonstrated by the presence of silver grains localizing incorporated radioactive uridine superimposed to these structures in which the presence of Us11 RNA and poly(A) tails was previously demonstrated.

Localization of the newly initiated and processed ribosomal primary transcripts during the mitotic cycle in Physarum polycephalum.

We analyzed the fate of the rRNA released as a result of the prophasic disintegration of the nucleolus, during the "closed" mitosis of the naturally synchronous plasmodium of Physarum polycephalum. Using a probe complementary to the mature 19S and 26S rRNA, we previously showed that the nucleolus-derived rRNAs are stable in mitosis, mainly associated with numerous fibrillar nucleolar remnants (Pierron and Puvion-Dutilleul, 1993, Exp. Cell Res.

Ultrastructure of the nucleus in relation to transcription and splicing: roles of perichromatin fibrils and interchromatin granules.

In the present article we summarize our results concerning the in situ ultrastructural organization of the nuclear steps of mRNA production using several cellular models including normal noninfected cells, herpes simplex virus type 1 and adenovirus type 5 infected cells, and transiently transfected cells. We confirm that perichromatin fibrils are the in situ morphological expression of nascent transcripts and the main support of splicing. Special emphasis is devoted to the clusters of interchromatin granules which seem to be involved in several central functions including accumulations of snRNP components as well as sorting and/or regulation of export of different RNA species.

Distribution of ribosomal genes in nucleoli of herpes simplex virus type 1 infected cells.

Herpes simplex virus type 1 (HSV-1) infection of HeLa cells induces profound changes in the structure of the nucleoli. They become markedly elongated, and their fibrillar centers become greatly diminished in number, but larger than in non-infected HeLa cells, and only partially surrounded by the dense fibrillar component. The effect of prolonged HSV-1 infection on the distribution of the rRNA genes was studied by means of postembedding electron microscope in situ hybridization using a biotinylated ribosomal DNA (rDNA) probe, which spans about half of the rRNA gene, and subsequent immunogold labeling of the resulting hybrids.

LYSP100-associated nuclear domains (LANDs): description of a new class of subnuclear structures and their relationship to PML nuclear bodies.

The PML gene is fused to the retinoic acid receptor alpha (RAR alpha) gene in t(15;17) acute promyelocytic leukemia (APL), creating a PML-RAR alpha fusion oncoprotein. The PML gene product has been localized to subnuclear dot-like structures variously termed PODs, ND10s, Kr bodies, or PML nuclear bodies (PML NBs). The present study describes the cloning of a lymphoid-restricted gene, LYSP100, that is homologous to another protein that localizes to PML NBs, SP100.

Nucleolin is a sequence-specific RNA-binding protein: characterization of targets on pre-ribosomal RNA.

Nucleolin is an abundant nucleolar protein, which plays an essential, but largely unknown role in ribosome biogenesis. Nucleolin contains four consensus RNA-binding domains (CS-RBD), the presence of which suggests that the molecular function of this protein is likely reflected by its RNA-binding properties. Indeed, by immunocytological analysis performed on ribosomal transcription units, we have found several nucleolin molecules associated with nascent pre-rRNA.

Non-isotopic electron microscope in situ hybridization for studying the functional sub-compartmentalization of the cell nucleus.

Post-embedding electron microscope in situ hybridization using gold particles as label permits the clear identification of the cellular structures which contain the nucleic acid molecules under study. It has yielded information on the distribution of defined nucleic acid sequences of different origins-cellular or viral, DNA or RNA, single- or double-stranded molecules-which has revolutionized the study of the nucleus. Application of this powerful technique in combination with other refined techniques to studies on transcription and replication of cellular and viral genes has augmented our knowledge of the functional organization of the cell nucleus.