Identification and characterization of mouse PSF1-binding protein, SLD5

Although most somatic cells cannot proliferate, immature cells proliferate continuously to produce mature cells. Recently, we cloned mouse PSF1 from a hematopoietic stem cell specific cDNA library and reported that PSF1 is indispensable for the proliferation of immature cells. To identify the PSF1-binding protein, we used the yeast two-hybrid system with PSF1 as bait, and identified and cloned SLD5. SLD5 interacted with a central region of PSF1. Tissue distribution of SLD5 was quite similar to that of PSF1. When overexpressed, SLD5 protein was co-localized with PSF1. These data suggest that PSF1 and SLD5 may cooperate in the proliferation of immature cell populations.     

Human cells lacking coilin and Cajal bodies are proficient in telomerase assembly,trafficking and telomere maintenance

The RNA component of human telomerase (hTR) localizes to Cajal bodies, and it has been proposed that Cajal bodies play a role in the assembly of telomerase holoenzyme and telomerase trafficking. Here, the role of Cajal bodies was examined in Human cells deficient of coilin (i.e. coilin-knockout (KO) cells), in which no Cajal bodies are detected. In coilin-KO cells, a normal level of telomerase activity is detected and interactions between core factors of holoenzyme are preserved, indicating that telomerase assembly occurs in the absence of Cajal bodies. Moreover, dispersed hTR aggregates and forms foci specifically during S and G2 phase in coilin-KO cells. Colocalization of these hTR foci with telomeres implies proper telomerase trafficking, independent of Cajal bodies. Therefore, telomerase adds similar numbers of TTAGGG repeats to telomeres in coilin-KO and controls cells. Overexpression of TPP1-OB-fold blocks cell cycle-dependent formation of hTR foci and inhibits telomere extension. These findings suggest that telomerase assembly, trafficking and extension occur with normal efficiency in Cajal bodies deficient human cells. Thus, Cajal bodies, as such, are not essential in these processes, although it remains possible that non-coilin components of Cajal bodies and/or telomere binding proteins (e.g. TPP1) do play roles in telomerase biogenesis and telomere homeostasis.     

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.     

Isolation and biochemical characterization of nuclei from immature and mature guinea pig granulocytes

Intact nuclei were isolated in high yield from enriched fractions of immature and mature guinea pig granulocytic leukocytes. These nuclei were used to determine whether any changes in synthesis and content of nuclear proteins accompany the striking increase in chromatin condensation and the nuclear lobation which occur during granulocyte maturation. The results indicate that the synthesis of nuclear proteins and the nuclear RNA content decrease markedly during granulocyte maturation. The incorporation of l-[U-¹⁴C]leucine into the acid-soluble histone-rich fraction of chromatin from immature cells is about 25 times that of mature cells, and the incorporation into the acid-insoluble, nonhistone proteins of chromatin from immature cells is about 6 times that of mature cells. It appears that there is very little quantitative change with respect to the protein components of nuclei from immature and mature granulocytic leukocytes. No significant differences in the amounts of histone, nonhistone protein, or phosphoprotein between nuclei of immature and mature granulocytes could be detected. No major differences in gel electrophoretic patterns of histones or nonhistone proteins could be detected. The fact that the amount of the chromatin proteins remains relatively constant during cell maturation in spite of the pronounced decrease in the rate of synthesis suggests that the rate of turnover of these proteins decreases significantly as the maturation of granulocytic leukocytes proceeds.     

Morphology of the reproductive system of the armadillo. The spermatogonia

Spermatogonia of the nine-banded armadillo, Dasypus novemcinctus mexicanus, were studied morphologically using light and electron microscopy and examined histochemically using light microscopy. Immature flat type spermatogonia have ovoid or irregular nuclei with loosely condensed chromatin. Free ribosomes are abundant while profiles of rough endoplasmic reticulum are scarce. Smooth endoplasmic reticulum is a prominent feature occasionally taking an unusual cylindrical form. Mature spermatogonia exhibit rounder nuclei with greater degrees of chromatin clumping. Smooth endoplasmic reticulum is no longer prominent whereas profiles of rough endoplasmic reticulum are quite common. Occasional lysosomal configurations are found in mature spermatogonia. The majority of spermatogonial cells exhibit weak to moderate reactivity when stained with the periodic acid-Schiff (PAS) reaction. Certain cells in each tubular cross section stain vividly with this reaction and the PAS positivity is removable with salivary amylase. Because of nuclear characteristics, position of the cell immediately upon the basal lamina, intensity of the PAS reaction and the relative paucity of the vividly staining cells, it is suggested that they are members of the immature spermatogonial cell line, perhaps acting as stem cells. None of the several other histochemical procedures employed was capable of selectively demonstrating these cells.     

Nuclear bodies in the oocyte nucleus of ground beetles are enriched in snRNPs

Within the oocyte nucleus of many insect species, a variable number of intensely stained spherical bodies occur. These nuclear bodies differ significantly from nucleoli and their precise role in nuclei has not been elucidated yet. I have examined some of the histochemical properties as well as the molecular composition of these structures in a representative of ground (carabid) beetles. I demonstrate, using molecular markers, that the nuclear bodies are composed of small nuclear RNAs and associated proteins, including p80 coilin. Hence, they correspond to Cajal bodies (= coiled bodies) described in somatic cell nuclei as well as oocyte germinal vesicles in plant and animal organisms. It is suggested that Cajal bodies in the carabid germinal vesicle serve as a storage site for splicing factors.     

In vivo analysis of Cajal body movement, separation, and joining in live human cells

Cajal bodies (also known as coiled bodies) are subnuclear organelles that contain specific nuclear antigens, including splicing small nuclear ribonucleoproteins (snRNPs) and a subset of nucleolar proteins. Cajal bodies are localized in the nucleoplasm and are often found at the nucleolar periphery. We have constructed a stable HeLa cell line, HeLa(GFP-coilin), that expresses the Cajal body marker protein, p80 coilin, fused to the green fluorescent protein (GFP-coilin). The localization pattern and biochemical properties of the GFP-coilin fusion protein are identical to the endogenous p80 coilin. Time-lapse recordings on 63 nuclei of HeLa(GFP-coilin) cells showed that all Cajal bodies move within the nucleoplasm. Movements included translocations through the nucleoplasm, joining of bodies to form larger structures, and separation of smaller bodies from larger Cajal bodies. Also, we observed Cajal bodies moving to and from nucleoli. The data suggest that there may be at least two classes of Cajal bodies that differ in their size, antigen composition, and dynamic behavior. The smaller size class shows more frequent and faster rates of movement, up to 0.9 microm/min. The GFP-coilin protein is dynamically associated with Cajal bodies as shown by changes in their fluorescence intensity over time. This study reveals an unexpectedly high level of movement and interactions of nuclear bodies in human cells and suggests that these movements may be driven, at least in part, by regulated mechanisms.     

A Cajal body-independent pathway for telomerase trafficking in mice

The intranuclear trafficking of human telomerase involves a dynamic interplay between multiple nuclear sites, most notably Cajal bodies and telomeres. Cajal bodies are proposed to serve as sites of telomerase maturation, storage, and assembly, as well as to function in the cell cycle-regulated delivery of telomerase to telomeres in human cells. Here, we find that telomerase RNA does not localize to Cajal bodies in mouse cells, and instead resides in separate nuclear foci throughout much of the cell cycle. However, as in humans, mouse telomerase RNA (mTR) localizes to subsets of telomeres specifically during S phase. The localization of mTR to telomeres in mouse cells does not require coilin-containing Cajal bodies, as mTR is found at telomeres at similar frequencies in cells from wild-type and coilin knockout mice. At the same time, we find that human TR localizes to Cajal bodies (as well as telomeres) in mouse cells, indicating that the distinct trafficking of mTR is attributable to an intrinsic property of the RNA (rather than a difference in the mouse cell environment such as the properties of mouse Cajal bodies). We also find that during S phase, mTR foci coalesce into short chains, with at least one of the conjoined mTR foci co-localizing with a telomere. These findings point to a novel, Cajal body-independent pathway for telomerase biogenesis and trafficking in mice.     

Profilin I colocalizes with speckles and Cajal bodies: a possible role in pre-mRNA splicing

Profilin is one of the major components controlling actin polymerization. Here, profilin I was located in fibroblasts and HeLa cells by the use of two different sets of affinity-purified antibodies. Both antibody preparations labeled nuclei in a speckle-like pattern and displayed extensive colocalization with small nuclear ribonucleoprotein particle (snRNP)-core proteins and p80 coilin-containing Cajal bodies. Treatment with actinomycin D led to largely similar reorganizations of snRNPs and profilin, while profilin and Cajal bodies separated under these conditions. One of the profilin antibodies interfered with pre-mRNA splicing in vitro, further indicating a role for profilin during pre-mRNA processing.     

Nuclear bodies in Douglas fir (Pseudotsuga menziesii Mirb.) microspores

The identification of nucleolar proteins and immunocytochemical localization of small nuclear ribonucleoprotein (snRNP) elements revealed the presence of three types of nuclear bodies in Douglas fir microspore nuclei. One type consists of structures resembling Cajal bodies (CBs) and contains nucleolar proteins as well as snRNPs and U2 snRNA. The second type is bizonal bodies, which are nuclear bodies also linked with the splicing system. The bizonal body comprises two parts: the first contains Sm proteins and stains strongly with silver stain, and the second resembles CBs in terms of the degree of silver staining and molecular composition. Douglas fir is the second species after larch where the presence of bizonal bodies has been demonstrated. Pseudotsuga menziesii Mirb and Larix decidua Mill are species with one of the longest microsporogenesis processes known in plants. The presence of bizonal bodies in both species may be linked to the intensification of the splicing processes in microspores with an exceptionally long cell cycle. The third type of structure is dense bodies, whose morphology and degree of silver staining strongly indicate their functional and spatial relationship to the dense part of bizonal bodies.     

Human granulocyte-specific nuclear antigen(s). II. Detection of antigens in human proliferative syndromes

Antisera raised to dehistonized chromatin from isolated normal human granulocytes revealed the presence of chromatin-associated antigens specific for the human neutrophils that appear during late stages of myeloid cellular differentiation. Immunological specificity was demonstrated by C fixation, immunodiffusion, and immunocytochemical reactions. Chromatin prepared from both normal granulocytes and specimens of myeloid leukemia showed immunologic reactivity. Although the normal antigens were detected in a specimen of CML, the position of immunodiffusion precipitin lines was different from that obtained with normal granulocyte chromatin. In addition, chromatin prepared from the myeloid leukemic cell line HL-60 expressed only one of the three precipitin bands normally found in immunodiffusion. The immunocytochemical staining reaction was confined to the nucleus of mature neutrophils in normal peripheral blood smears. Greater than 90% of cells in peripheral blood specimens of CML showed positive immunocytochemical nuclear staining. In other types of leukaemia, the normal mature granulocyte reacted with antiserum, but the nonmyeloid leukemic cells in these specimens did not. The specificity of immunologic reactions described here suggests the usefulness of nuclear antigens as cell markers.     

Stage-Specific Expression and Localization of MENT, a Nuclear Protein Associated with Chromatin Condensation in Terminally Differentiating Avian Erythroid Cells

Polyclonal antibodies have been raised against a nonhistone protein (MENT) which has been previously shown to be associated with the repressed chromatin of mature chicken erythrocytes and to promote the in vitro condensation of chromatin of immature erythrocyte nuclei. Here we report that the expression pattern of MENT closely follows chromatin condensation in maturing arian erythrocytes of definitive and primary lineages. Accumulation of MENT correlates more strongly with chromatin condensation than does accumulation of histone H5. In addition to being present in erythrocytes, the protein was also found in neutrophil nuclei and an immunofluorescence reaction was observed with embryonic (nucleated) thrombocytes. MENT was not detected in other chicken tissues (brain, liver, testis). In intact erythrocytes, MENT immunofluorescence was found in foci close to the nuclear periphery, while in isolated, decondensed nuclei, the fluorescence signal was uniformly distributed. In neutrophil nuclei, containing approximately 10 times more MENT than adult erythrocytes, intense staining associated with the peripheral heterochromatin was observed. These findings are discussed in regard to a possible mechanism for chromatin condensation by MENT.     

Discrepancies between cytophotometric alkaline Fast Green measurements and nuclear histone protein content

Synopsis Nuclei of different cell types of the myelopoietic series from normal human bone marrow smears were stained with Fast Green FCF, and their dye uptake estimated by cytophotometry. In very immature cell types (myeloblasts and promyelocytes), a high percentage of nuclei either did not stain, or had a dye content too low to be measured. Fast Green absorbencies were increased in the more mature stages. The highest values occurred in mature polymophonuclear leukocytes. The varying Fast Green absorbancies in the nuclei of different cell types suggest that the staining capacity of histone proteins depends on the functional state of the chromatin and does not indicate variations in the histone content of the nuclei.     

A feeder-free hematopoietic differentiation system with generation of functional neutrophils from feeder- and cytokine-free primate embryonic stem cells

We have established a novel feeder- and recombinant cytokine-free culture system for the maintenance of primate embryonic stem (ES) cells along with a feeder-free hematopoietic differentiation protocol for high efficiency CD45-positive cell production. In our system, cynomolgus monkey ES cells were properly maintained in an undifferentiated state with high immature marker expressions and teratoma-producing activities. Embryoid bodies (EBs) were generated in the presence of serum and cytokine cocktail and subjected to attachment culture on gelatin-coated plates. After about 2 weeks, a sac-like structure filled with abundant round cells emerged at the center of flattened EB. Then total cells were collected and transferred onto new gelatin-coated plates, where cells were firmly attached and actively proliferated to confluence. After another few days culture, abundant floating cells were detected in the culture supernatant. These cells expressed high levels of CD45 (>90%), while adherent cells expressed low levels of CD45 (<10%). The former consisted of various differentiated stages of myeloid cells from immature myeloblasts to mature polymorphonuclear neutrophils and macrophages. Although the percentages of neutrophils varied between 10 to 20 depending on experiments, their mature phenotype was reproducibly confirmed by specific staining and functional assays. Our protocol provides the minimum essence for primate ES cell maintenance and hematopoietic differentiation that is beneficial from economical and clinical points of view.     

Beyond Repair Foci: Subnuclear Domains and the Cellular Response to DNA Damage

In the mid to late 1990's several groups identified DNA damage-dependent focal accumulations in nuclei of both DNA repair factors and the phosphorylated form of the histone variant H2A.X. The term "repair foci" has since been used to describe these protein accumulations. As a molecular marker for DNA damage, they have been immensely useful in the study of signal transduction pathways triggered by DNA damage while aiding in the identification of new factors involved in DNA repair. In spite of their importance, many other changes in the nuclear landscape correlate with DNA damage and repair processes. These include dramatic changes in chromatin ultrastructure and epigenetic modifications, which occur at the site of DNA breaks as well as globally throughout the nucleus. Besides chromatin, DNA damage also affects the dynamic behaviour, morphology and biochemical composition of various subnuclear domains, including the nucleolus, promyelocytic leukemia (PML) nuclear bodies and Cajal bodies. These changes in the nuclear landscape, the topic of this review, appear to be intimately linked to the cellular response to DNA damage and may prove as useful as repair foci in elucidating mechanisms of DNA repair.