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N Ringertz

Publications and source records attributed to N Ringertz.

At least 19 recordsLinked to original sources
PubMed1998

Intracellular localisation of the Ro 52kD auto-antigen in HeLa cells visualised with green fluorescent protein chimeras.

Autoantibodies to the Ro/SSA and La/SSB antigens are found in patients with Sjogren's syndrome and systemic lupus erythematosus. The Ro/SSA autoantigen consists of a 52 kD and a 60 kD protein, complexed with one of four small RNA molecules. The La protein can associate with the complex. The Ro/SSA autoantigens are present in all mammalian cells, but their intracellular location is subject of controversy and their function remains unclear. To study the intracellular sorting and targeting of Ro 52 kD we have constructed expression plasmids encoding fusion proteins between the full-length Ro 52 kD protein as well as Ro 52 kD fragments and the green fluorescent protein (GFP) from the jelly fish, Aequorea Victoria. The subcellular distribution of the GFP-Ro 52 kD fusion proteins was investigated in transient expression experiments using transfected HeLa cells. The GFP-full-length Ro 52 kD fusion protein was accumulated in the cytoplasm and excluded from the nucleus. When GFP was fused with the La protein, the fluorescence was located in the nucleus. Clones coding for Ro 52 kD fragments containing the hydrophilic central part of the Ro 52 kD protein gave the same intracellular location and type of cytoplasmic speckles as the full-length Ro 52 kD protein. In contrast, both amino terminal and carboxy terminal fragments were uniformly distributed throughout the cell just like the GFP protein itself. These observations indicated a cytoplasmic location of the Ro 52 kD protein and demonstrated the crucial role of the hydrophilic domain in restricting the Ro 52 kD protein to this intracellular compartment.

Animals↗
PubMed1997

Intracellular distribution of an integral nuclear pore membrane protein fused to green fluorescent protein--localization of a targeting domain.

The 121-kDa pore membrane protein (POM121) is a bitopic integral membrane protein specifically located in the pore membrane domain of the nuclear envelope with its short N-terminal tail exposed on the luminal side and its major C-terminal portion adjoining the nuclear pore complex. In order to locate a signal for targeting of POM121 to the nuclear pores, we overexpressed selected regions of POM121 alone or fused to the green fluorescent protein (GFP) in transiently transfected COS-1 cells or in a stably transfected neuroblastoma cell line. Microscopic analysis of the GFP fluorescence or immunostaining was used to determine the intracellular distribution of the overexpressed proteins. The endofluorescent GFP tag had no effect on the distribution of POM121, since the chimerical POM121-GFP fusion protein was correctly targeted to the nuclear pores of both COS-1 cells and neuroblastoma cells. Based on the differentiated intracellular sorting of the POM121 variants, we conclude that the first 128 amino acids of POM121 contains signals for targeting to the continuous endoplasmic reticulum/nuclear envelope membrane system but not specifically to the nuclear pores and that a specific nuclear pore targeting signal is located between amino acids 129 and 618 in the endoplasmically exposed portion of POM121.

Animals↗
PubMed1997

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Journal Article↗
PubMed1997

Effects of divalent cations on M-cadherin expression and distribution during primary rat myogenesis in vitro.

In the process of myogenesis, cadherins are thought to be involved in the initial cell-cell recognition and possible initiation of myoblast fusion to form multinucleated myotubes. Of the cadherins, M-cadherin, but not N-cadherin, is down-regulated upon inhibition of myogenesis, suggesting that M-cadherin may be a key receptor involved in myogenesis. M-cadherin binds in a calcium-dependent manner, and depletion of divalent cations inhibits myoblast fusion. We analyzed the regulation of M-cadherin protein and mRNA levels in primary rat myogenic cultures in the presence and absence of divalent cations. In untreated cultures M-cadherin was localized to various myogenic cell-cell contacts. M-cadherin protein and mRNA levels showed a peak at day 2 after the initiation of growth. When divalent cations were removed from the cell culture medium, myoblast fusion was inhibited and immunocytochemical analysis revealed a failure of M-cadherin to localize to cell-cell contacts. Analysis of M-cadherin protein and mRNA in fusion-inhibited cultures still revealed a peak at day 2. However, by day 3, M-cadherin protein levels in the fusion-inhibited cultures were reduced in both the detergent-soluble and -insoluble fractions in comparison with the untreated cultures. Interestingly, beta-catenin, a protein associated with cadherins, was frequently observed at intercellular contacts in the fusion-inhibited cultures. We could also show that the intracellular levels of beta-catenin protein remained constant regardless of the presence or absence of divalent cations. In summary, the dynamic regulation of M-cadherin in muscle-fusion-related events is an indication of the importance of M-cadherin for myoblast fusion and myogenic differentiation.

Animals↗
PubMed1997

Altered distribution of the promyelocytic leukemia-associated protein is associated with cellular senescence.

The disruption of the normal function and nuclear localization of the promyelocytic leukemia-associated protein (PML) may play a major role in the pathogenesis of acute promyelocytic leukemia. PML, which is concentrated in nuclear bodies (PML bodies), has been shown to have growth- and transformation-suppressive properties. In this study, we have examined the intranuclear distribution of PML in a conditionally immortalized human cell line (IDH4) in which both proliferation and immortalization are dependent on the presence of SV40-encoded large T-antigen (SV40T). Expression of SV40T is controlled by a dexamethasone (Dex)-inducible promotor. Suppression of SV40DT (Dex removal) in IDH4 cells causes G1 arrest and expression of the senescent phenotype. This is accompanied by a redistribution of PML in most cells from the usual pattern containing only spherical bodies to a pattern, containing large doughnut-like or fiber-like structures in addition to the spherical bodies. This change in pattern is reversed when phenotypically senescent IDH4 cells are stimulated to proliferate again by SV40T-induction. Moreover, we find that there is a similar change in the PML pattern between young and senescent or serum-starved young IMR90 human fibroblasts, from which IDH4 cells are derived. However, fewer serum-starved cells contain large PML bodies than senescent cells. Our observations suggest senescence, although it may be partly related to growth arrest. Using three-dimensional fluorescence digital imaging microscopy, we have found that the apparently doughnut-like PML structures have a cylindrical or egg-shaped form and that PML is concentrated to the outer shell of the structure.

Cell Division↗
PubMed1996

Intranuclear redistribution of SV40T, p53, and PML in a conditionally SV40T-immortalized cell line.

We have previously reported that EBNA-5, one of the Epstein-Barr virus-encoded proteins, accumulates in the nuclear bodies containing PML, the promyelocytic leukemia associated protein. In this study, we examine the intranuclear distribution of SV40 large T-antigen (SV40T), the p53 tumor suppressor protein (p53), and PML in a conditionally immortalized cell line, IDH4. In IDH4 cells, the expression of SV40T is regulated by a dexamethasone (Dex)-driven promoter. Withdrawal of Dex results in down-regulation of SV40T and growth arrest, whereas addition of Dex to the growth-arrested cells results in up-regulation of SV40T and proliferation. In proliferating IDH4 cells, SV40T is concentrated in nuclear dots that are also positive for p53. Many of these dots are juxtaposed to PML positive structures but do not colocalize with them. After removal of Dex, SV40T-p53 dots gradually disappear, while the PML structures remain. Induction of SV40T in nonproliferating IDH4 cells causes a coordinated redistribution of SV40T and p53. The immunostaining for SV40T and p53 is first weak, then strong with a homogeneous distribution, and 3-4 days later becomes dot-like again. This reappearance of SV40T-p53 dots coincides with the recovery of proliferation in restimulated IDH4 cells. Also, the p53 pattern correlates with the SV40T pattern with regard to both morphology and intensity during both suppression and induction of SV40T. Taken together, our data suggest that (i) the level of p53 is coregulated with the level of SV40T in a dose-dependent fashion; (ii) the formation of SV40T-p53 nuclear dots correlates with the transformed phenotype; (iii) the SV40T-p53 dots localize preferentially to the neighborhood of PML bodies which are already present in normal cells.

Antigens, Polyomavirus Transforming↗
PubMed1996

Formation of nuclear bodies in cells overexpressing the nuclear pore protein POM121.

POM121 is an integral membrane protein specifically localized in the pore membrane domain of the nuclear envelope. We have investigated the intracellular distribution of rat POM121 heterologously overexpressed in monkey COS cells by immunofluorescence and fluorescence digital imaging microscopy. At low levels of expression overexpressed POM121 was distributed in the nuclear envelope in a punctate fashion, partially overlapping with the distribution of nuclear pores. At high levels of expression, however, the overexpressed protein accumulated in intranuclear bodies. These bodies represent a novel subnuclear structure, displaying a defined cylindrical structure and a distinct localization at or adjacent to the inner nuclear membrane. The C-terminal portion of POM121, which contains a pentapeptide repeat domain common to a subfamily of related nucleoporins, was sufficient to mediate targeting to the nuclear envelope as well as formation of intranuclear bodies.

Animals↗
PubMed1996

The Epstein-Barr virus-encoded nuclear antigen EBNA-5 accumulates in PML-containing bodies.

EBNA-5 is one of the Epstein-Barr virus (EBV)-encoded nuclear proteins required for immortalization of human B lymphocytes. In the nuclei of EBV-transformed lymphoblastoid cell lines EBNA-5 is preferentially targetted to distinct nuclear foci. Previously we have shown (W.Q. Jiang, L. Szekely, V. Wendel-Hansen, N. Ringertz, G. Klein, and A. Rosen, Exp. Cell Res. 197:314-318, 1991) that the same foci also contained the retinoblastoma (Rb) protein. Using a similar double immunofluorescence technique, we now show that these foci colocalize with nuclear bodies positive for PML, the promyelocytic leukemia-associated protein. Artificial spreading of the chromatin by exposure to the forces of fluid surface tension disrupts this colocalization gradually, suggesting that the bodies consist of at least two subcomponents. Heat shock or metabolic stress induced by high cell density leads to the release of EBNA-5 from the PML-positive nuclear bodies and induces it to translocate to the nucleoli. In addition to their presence in nuclear bodies, both proteins are occasionally present in nuclear aggregates and doughnut-like structures in which PML is concentrated in an outer shell. Nuclear bodies with prominent PML staining are seen in resting B lymphocytes. This staining pattern does not change upon EBV infection. In freshly infected cells EBNA-5 antigens are first distributed throughout the nucleoplasm. After a few days intensely staining foci develop. These foci coincide with PML-positive nuclear bodies. At a later stage and in established lymphoblastoid cell lines EBNA-5 is almost exclusively present in the PML-positive nuclear foci. The colocalization is restricted to EBV-infected human lymphoblasts. The data presented indicate that the distinct EBNA-5 foci are not newly formed structures but the result of translocation of the viral protein to a specialized domain present already in the nuclei of uninfected cells.

Antigens, Viral↗
PubMed1995

Resting B-cells, EBV-infected B-blasts and established lymphoblastoid cell lines differ in their Rb, p53 and EBNA-5 expression patterns.

Using immunofluorescence technique we have analysed the Rb, p53, EBNA-2 and EBNA-5 expression pattern in EBV infected human B-cells and established lymphoblastoid cell lines (LCL-s). Resting B-cells showed only a faint Rb and no p53 immunostaining. The expression of both Rb and p53 increased after EBV infection. The change was first detectable 6 h after infection. The frequency of brilliantly Rb positive cells increased more rapidly than p53 positives. EBNA-2 and EBNA-5 became first detectable 12 h after infection. The frequency of EBNA positive cells in the freshly infected cultures was concordant with the proportion of CD23 and PCNA positives, but remained consistently below the frequency of Rb and p53 positive cells. Double immunofluorescence staining showed that all EBNA-5 positive cells were strongly Rb and p53 positive. LCL-s did not stain for p53, whereas the Rb staining was maintained at a high level. The EBNA-5 staining pattern changed from brilliant almost homogeneous nuclear staining in the freshly infected B-cells, to a nonhomogeneous pattern with a small number of strongly fluorescent nuclear bodies in established LCL-s. There was no change in the EBNA-2 staining pattern. Our findings indicate that the immortalization of B-cells by EBV may initially involve a high expression of EBNA-5, p53 and Rb, but only cells with low p53 and focal expression of EBNA-5 in nuclear bodies have the selective advantage required to grow into immortalized lines.

Antigens, Viral↗
PubMed1995

Reversible nucleolar translocation of Epstein-Barr virus-encoded EBNA-5 and hsp70 proteins after exposure to heat shock or cell density congestion.

The Epstein-Barr virus (EBV)-encoded, nuclear matrix-associated EBNA-5 protein is preferentially localized within distinct nuclear blobs in EBV-immortalized lymphoblastoid cell lines. We have previously found that the same blobs also contain retinoblastoma (Rb) protein. We now show that they contain hsp70 protein as well. Both EBNA-5 and hsp70 translocate to the nucleolus under cell density congestion or after heat shock. Both proteins relocate to their original position upon the re-establishment of normal physiological conditions. EBNA-5 is tightly bound to the nuclear matrix. The translocated EBNA-5 is also tightly associated with matrix structures, as shown by sequential elution-based cell fractionation. The Rb protein does not translocate to the nucleolus. The virally encoded EBNA-1, -2, -3 and -6, and cellular PCNA, snRNP and cyclin E are not affected either. The translocation of EBNA-5 to the nucleolus is not species- or cell type-specific since stress conditions induced the same phenomenon in EBNA-5-transfected human, mouse and rat cells of different tissue origins.

Animals↗
PubMed1993

Position-dependent nuclear accumulation of the retinoblastoma (RB) protein during in vitro myogenesis.

The expression of the retinoblastoma (RB) protein has been studied during in vitro muscle differentiation by immunofluorescence staining with three different antibodies against RB protein. Proliferating mononucleate L6 rat myoblasts showed a low level of expression. As cells began to enter a nonreplicating G0 state, the cell population became heterogeneous. Some nonreplicating cells showed a high level of expression. Nuclei at the two ends of myotubes were strongly positive, whereas centrally located nuclei showed low RB expression. Overexpression of the human RB protein in rat L6 myotubes from a Semliki forest virus (SFV)-based, transient expression vector produced a similar picture. Terminally located nuclei expressed human RB at a much higher level than did the centrally located nuclei. The results suggest that individual nuclei with a multinucleated syncytium may undergo position-dependent specialization.

Animals↗
PubMed1992

Cell type and differentiation dependent heterogeneity in retinoblastoma protein expression in SCID mouse fetuses.

The expression pattern of retinoblastoma (Rb) protein has been studied at the single cell level in frozen sections of 16- to 18-day-old SCID mouse fetuses by immunofluorescence staining with mouse monoclonal and rabbit polyclonal antibodies, using conventional epifluorescence and confocal laser scanning microscopy. The nuclei of megakaryocytes, hemopoietic islands of the fetal liver, osteo-, amelo-, and odontoblasts, and skeletal muscle were strongly stained. There was no detectable Rb staining in the basal cell layers of stratified squamous epithelia, but the differentiating, more superficial layers were positive. Intestinal crypts were negative, whereas the villi were positive. In the retina, Rb protein was detectable in the inner ganglion layer but not in the outer neuroblastic layer. In the central nervous system, Rb protein was present in neurons and glia cells as well. The nuclei in the collecting tubules of the kidney, the pancreas, and the adrenal cortex were Rb positive. Analysis of the differentiation dependent expression of Rb protein in relation to the prospective life cycle of the cells in which it appears may pave the way toward an understanding of the tissue specific oncogenic effect of Rb loss in families with hereditary retinoblastoma.

Animals↗
PubMed1991

Intranuclear localization of a new snRNP-related antigen.

The intranuclear distribution of a new antigen (F78) associated with U snRNPs (small nuclear RNA-protein complexes) was compared with that of the RNP and Sm protein antigens previously identified on individual snRNP particles. Human and rat cells were double stained with human autoantisera and mouse monoclonal antibodies. The binding of the human and mouse antibodies was detected with secondary antibodies conjugated with fluorescein and rhodamine, respectively. The resulting immunofluorescence patterns were compared by digital image analysis. The F78, RNP, and Sm antigens show speckled fluorescence patterns which overlap to a great extent. The F78 pattern, however, also contains two classes of structural elements not present in the RNP pattern. Furthermore, during mitosis expression of the F78 antigen is completely suppressed from early prophase to telophase, while the RNP and Sm antigens are found evenly distributed throughout the cytoplasm of the dividing cells.

Animals↗
PubMed1991

Co-localization of the retinoblastoma protein and the Epstein-Barr virus-encoded nuclear antigen EBNA-5.

A monoclonal antibody (aRB1C1) raised against an Rb fusion protein detects a limited number (4-10) of relatively large intranuclear foci in an EBV-immortalized cord blood cell line (IB4). These domains also bind an anti-EBNA-5 monoclonal antibody. The Rb antibody reactive sites also co-localize with the SV40 T antigen in transformed monkey cells (COS). The nuclear structures stained by aRB1C1 and EBNA-5 antibodies are distinct from the structures detected with antibodies against centromeric proteins and certain snRNP epitopes. EBNA-5/Rb-positive domains do not selectively react with antibodies against the La antigen known to associate with the small EBV-encoded nuclear RNA species designated as the EBERs.

Antibodies, Monoclonal↗
PubMed1991

Intranuclear distribution of Epstein-Barr virus-encoded nuclear antigens EBNA-1, -2, -3 and -5.

Epstein-Barr virus (EBV)-transformed lymphoblastoid cell lines (LCLs) express at least seven virally encoded proteins. Their functional role, and their relationships to each other and to normal nuclear constituents are virtually unknown. As the first step towards a topographical study, the intranuclear distribution of EBV-encoded nuclear antigens EBNA-1, -2, -3 and -5 (abbreviated E1, E2 etc.) was examined in EBV-transformed LCLs by immunofluorescence and digital image analysis of fluorescence patterns. E1-E3 showed a finely granular distribution. The E2 patterns were virtually identical when comparing indirect staining using an E2-specific mouse monoclonal antibody with anticomplement immunofluorescence using a human antibody, rendered monospecific to E2 by absorption. The E1/E2 patterns showed 32% overlap and the E2/E3 10% overlap in the high overlap category (66.7-100%), while the E2/E2 comparison with two reagents showed 61% overlap in this category. This suggests that E2 and E3 largely appear in different nuclear structures, whereas E1 appears to be randomly distributed with regard to E2. The E5 pattern was radically different from that of E1, E2 and E3. The anti-E5 mouse monoclonal antibody detected 4-10 huge, globular, sharply circumscribed dots, located in dispersed chromatin areas, while the distribution of E1, E2 and E3 showed no obvious relationship to chromatin distribution. The methods described here allow a more refined topographical analysis of the EBNA protein family, mostly in relation to each other, in relation to other nuclear proteins, and with respect to specialized functional domains in interphase chromatin.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1990

Appearance and origin of snRNP antigens in chick erythrocyte nuclei reactivated in heterokaryons.

Fusion of terminally differentiated chick erythrocytes (CE) with transcriptionally active rat myoblasts results in heterokaryons in which the CE nuclei undergo reactivation of RNA synthesis and splicing. In order to analyze the transport and assembly of small nuclear ribonucleoprotein (snRNP) particles and larger molecular complexes engaged in RNA processing, we have examined CE nuclei in heterokaryons for the presence of four U snRNP-related nuclear antigens (Sm, 70,000 Mr, F78 and M3G-cap) and for one antigen (La), associated with RNA polymerase III transcripts. Inactive erythrocyte nuclei showed low levels of Sm and F78 antigens, but the other antigens were undetectable. Immediately after fusion, the fluorescence of the pre-existing chicken Sm antigen was detected in the CEn, and then the intensity of the signal increased rapidly during reactivation. The other antigens appeared more slowly, reaching full intensity at different time points after fusion. Blocking of chick transcription did not block the appearance of Sm, 70,000 Mr, cap and La antigens but did effectively inhibit the appearance of the F78 antigen. It has previously been demonstrated that the structure recognized by this monoclonal antibody is physically associated with functional splicing complexes. Blocking of translation in heterokaryons abolished uptake of snRNPs into the chicken nuclei. Taken together, the results indicate that rat snRNP complexes were imported into the chick nuclei after assembly in the cytoplasm. For all the studied antigens, except F78, this translocation was independent of chick RNA synthesis. The appearance of the F78 antigen was totally dependent on expression of chicken genes.

Animals↗