Chromatin structure in situ: the contribution of DNA ultrastructural cytochemistry.

Ultrastructural studies conducted in situ using conventional transmission electron microscopy have had relatively little impact on defining the structural organization of chromatin. This is due to the fact that in routine transmission electron microscopy, together with the deoxyribonucleoprotein, many different intermingled substances are contrasted, masking the ultrastructure of chromatin. By selective staining of DNA in thin sections, using the Feulgen-like osmium-ammine reaction, these drawbacks have been overcome and worthwhile data have been obtained both on the gross morphology and the ultrastructural-functional organization of chromatin in situ. In the present study these results are reviewed and discussed in light of recent achievements in both interphase nuclear chromatin compartmentalization in interphase nuclei and in the structural organization of chromatin fibers in transcriptionally active and inactive chromatin.

Nuclear envelope and chromatin compositional differences comparing undifferentiated and retinoic acid- and phorbol ester-treated HL-60 cells.

The human leukemic cell line (HL-60) can be induced to differentiate in vitro to granulocytic form with retinoic acid (RA), or to monocytic/macrophage form with phorbol ester (TPA). The granulocytic form acquires nuclear lobulation, nuclear envelope-limited chromatin sheets (ELCS), and cytoskeletal polarization, none of which are acquired following treatment with TPA. Immunoblotting analyses and capillary zone electrophoresis demonstrated that following RA treatment: lamins A/C and B1, and vimentin decreased to negligible amounts; LAP2 beta, lamin B2 and emerin remained essentially unchanged; lamin B receptor (LBR) increased markedly; histone subtypes H1.4 and 1.5 exhibited dephosphorylation. Following TPA treatment: lamins A/C and B1, B2 and vimentin increased in amount; LAP2 beta and emerin remained essentially unchanged; LBR increased markedly; histone subtypes H1.4 and 1.5 exhibited dephosphorylation. Emerin, which was cytoplasmic in undifferentiated or granulocytic cells, localized into the nuclear envelope following TPA. Normal human granulocytes revealed compositional differences compared to granulocytic forms of HL-60, namely increased vimentin and appearance of histone subtype H1.3. A working hypothesis for nuclear lobulation postulates a combination of: increased nuclear envelope deformability due to lamins A/C and B1 deficiency; an increase in nuclear surface area/volume; an increase in chromatin-nuclear envelope interactions.

Retinoic acid differentiation of HL-60 cells promotes cytoskeletal polarization.

Retinoic acid (RA) treatment of HL-60 cells in vitro induces granulocytic differentiation, involving reorganization of the nucleus and cytoplasm, development of chemoattractant-directed migration, and eventual apoptosis. The present studies with HL-60/S4 cells document that major elements of the cytoskeleton are changed: actin increases by 50%; vimentin decreases by more than 95%. The cellular content of alpha-tubulin does not significantly change; but the centrosomal-microtubule (MT) array moves away from the lobulating nucleus. Cytoskeletal-modifying chemicals modulate this polarized reorganization: Taxol and cytochalasin D enhance centrosome movement; nocodazole reverses it. Cytoskeletal-modifying chemicals do not appear to affect nuclear lobulation or the integrity of envelope-limited chromatin sheets (ELCS). Employing bcl-2-overexpressing HL-60 cells permitted demonstration of nuclear lobulation, ELCS formation, and centrosome-MT movement concomitantly during RA-induced differentiation, implying independence between the cellular reorganization and apoptotic programs. RA appears to promote an inherent potential in HL-60 cells for cytoskeletal polarization, likely to be important for chemoattractant-directed cell migration, an established characteristic of mature granulocytes.

Retinoic acid induction of nuclear envelope-limited chromatin sheets in HL-60.

Exposure of the human leukemic cell line (HL-60) to 1 microM retinoic acid (RA) induces in vitro granulopoiesis, including the development of lobulated nuclei. Ultrastructural studies, presented here, demonstrate the formation of extensive quantities of nuclear envelope-limited chromatin sheets (ELCS), in addition to nuclear lobulation, following treatment with RA. ELCS contain DNA, as shown by the Feulgen-like electron microscope stain osmium ammine-B. Lamin B was demonstrated in ELCS by immunoelectron microscopy with colloidal gold-labeled antibody. Formation of ELCS occurred in Bcl2-overexpressing HL-60 cell sublines with suppressed apoptotic cell death, indicating separable mechanisms for ELCS formation and apoptosis. Immunofluorescent and immunoblotting procedures demonstrated modulations in the amounts and distribution of nuclear envelope-associated components. Total amounts of lamins A/C and cytoplasmic vimentin were reduced by RA treatment. The amounts of lamin B, lamin B receptor (LBR), and lamina-associated polypeptide 2 (LAP2) did not exhibit significant quantitative changes, but acquired heterogeneous staining patterns on the nuclear envelope. RA induced the appearance of low-molecular-weight LBR-related proteins. This study demonstrated the parallel induction of lobulated nuclei and of ELCS and the modulation of nuclear envelope components following exposure of HL-60 to retinoic acid.

Parental alleles of an imprinted mouse transgene replicate synchronously.

Molecular features of imprinted genes include differences in expression, methylation, and the timing of DNA replication between parental alleles. Whereas methylation differences always seem to be associated with differences in expression, differences in the timing of replication between parental homologs are not always seen at imprinted loci. These observations raise the possibility that differences in replication timing may not be an essential feature underlying genomic imprinting. In this study, we examined the timing of replication of the two alleles of the imprinted RSVIgmyc transgene in individual embryonic cells using fluorescence in situ hybridization (FISH). The cis-acting signals for RSVIgmyc imprinting are within RSVIgmyc itself. Thus, allele-specific differences in replication, if they indeed govern RSVIgmyc imprinting, should be found in RSVIgmyc sequences. We found that the parental alleles of RSVIgmyc, which exhibit differences in methylation, replicated at the same time. Synchronous replication was also seen in embryonic cells containing a modified version of RSVIgmyc that exhibited parental allele differences in both methylation and expression. These findings indicate that maintenance of expression and methylation differences between alleles does not require a difference in replication timing. The differences in replication timing of endogenous imprinted alleles detected by FISH might therefore reflect structural differences between the two alleles that could be a consequence of imprinting or, alternatively, could be unrelated to imprinting.

Osmium ammine-B and electron spectroscopic imaging of ribonucleoproteins: correlation of stain and phosphorus.

Ultra-thin sections of Chironomus salivary glands were stained in a non-Feulgen procedure with osmium ammine-B and imaged at several electron energy-loss windows. For two types of RNP-containing structures (ie Balbiani ring granules and endoplasmic reticulum), a significant spatial correlation was observed between stain distribution and net phosphorus distribution. Non-Feulgen osmium ammine-B staining does not require the use of ultra-thin sections and can approximate the distribution of nucleic acid phosphorus.

Modeling the 3-D RNA distribution in the Balbiani ring granule.

Mature Balbiani Ring (BR) granules in situ were stained with the nucleic acid specific stain, osmium ammine-B, recorded by electron spectroscopic imaging and reconstructed by electron microscope tomography to examine the three-dimensional (3-D) distribution of BR heterogeneous nuclear RNA (hnRNA). The BR2 granules contain ca. 37 kb of mRNA. Reconstructed BR granules were selected to emphasize one of the prevalent conformations seen in the sectioned salivary glands, the en face or "pin-wheel" conformation. A variety of image processing and volume-rendering operations were applied to the set of reconstructed BR granules. Some of the conclusions of this study are the following: (1) RNA distribution is not uniform throughout the granule; (2) RNA is condensed into about ten particles per granule, which all appear to possess approximately the same RNA stain density; (3) heterogeneity exists in the positions and sizes of particles within the various BR granules. These data argue for the folding of a beaded ribbon, consisting of connected particulate condensations of BR mRNA, possessing considerable 3-D flexibility, even in the packaged state. A comparison of this beaded-ribbon model and a prior folded hnRNP fiber model is also presented.

Inaccessibility of the Euplotes telomere binding protein.

The telomere binding protein (TP) from the macronucleus of the ciliate Euplotes eurystomus was purified by removal of tenaciously bound DNA with hydroxylapatite, and the purified TP partially sequenced. Rabbit antiserum was generated against a synthetic peptide of 14 amino acids at the amino-terminus of the TP. This antiserum was employed to examine the accessibility of TP antigenic determinants in nuclei and chromatin. Immunofluorescent staining of isolated macronuclei revealed only weak reactivity with specific antiserum. Reactivity within replication bands was demonstrated, and could be augmented by preparation of nuclear scaffolds. Employing a dot immunoblot analysis, the amino-terminal antigenic determinants of TP were revealed after extraction of histone H1 (and some nonhistones). A different aspect of TP inaccessibility was demonstrated by immunoblot analysis of trypsin-treated macronuclei and chromatin; TP was considerably less susceptible to digestion by trypsin than were histones H1 and H3. The relative inaccessibility of TP was not a consequence of chromatin higher-order structure, since soluble macronuclear chromatin in low salt exhibited the same burying of antigenic determinants by dot blot analysis, and the same decreased susceptibility to trypsin, as did isolated nuclei. Electron microscopy of soluble macronuclear chromatin spread in low salt revealed that most telomeres appear unfolded, without stable higher-order structure. The mechanisms for the relative inaccessibility of TP are not yet known, but probably arise as a consequence of the strong interactions of TP with the telomere nucleotide sequence and additional interactions of TP with various chromatin proteins, perhaps including histone H1.

Electron microscope tomography of Balbiani Ring hnRNP substructure.

Three-dimensional (3-D) reconstructions, by electron microscope tomography, of selectively stained, contrast enhanced Balbiani Ring (BR) hnRNP granules reveal a complex spatial arrangement of RNA-rich domains. This particulate substructure was examined by volume rendering computer graphics. Modeling the arrangement of RNA-rich domains is made difficult by apparent structural flexibility and/or heterogeneity of composition. Formulation of a consensus 3-D arrangement of RNA-rich domains will require an expanded data base of reconstructed BR granules and the development of new image manipulation and analysis techniques. This study demonstrates the potential for ultrastructural cell biology of combining several new techniques: selective nucleic acid staining, electron spectroscopic imaging to enhance contrast, electron microscope tomography and volume rendering computer graphics.

Balbiani ring hnRNP substructure visualized by selective staining and electron spectroscopic imaging.

The Balbiani Rings (BR) in the polytene chromosomes of Chironomus salivary glands are intense sites of transcription. The nascent RNPs fold during transcription into 40-50-nm granules, containing in the mature transcript approximately 37-kb RNA. Using a new nucleic acid specific stain, osmium ammine B on Lowicryl sections, in combination with electron energy filtered imaging of sections containing BR granules, we demonstrate a RNA-rich particulate substructure (10-nm particle diameter; 10-12 particles per BR granule). Elemental imaging supports that these particles are enriched in phosphorus. The possible relationship of these RNA-rich particles to ribonucleosomes is discussed, as well as models for their arrangement in the mature BR granules.

Proliferating cell nuclear antigen/cyclin in the ciliate Euplotes eurystomus: localization in the replication band and in micronuclei.

Human autoimmune sera specific for proliferating cell nuclear antigen (PCNA)/cyclin (auxiliary protein for DNA polymerase delta) demonstrated the presence of epitopes within the macro- and micronuclei of the hypotrichous ciliated protozoa Euplotes eurystomus. Tightly bound PCNA/cyclin was localized at the site of DNA synthesis in macronuclei, the rear zone of the replication band. Starvation or heat shock, conditions that reduce macronuclear replication, resulted in a decrease of PCNA/cyclin in replication bands. Micronuclei also exhibited PCNA/cyclin localization which persisted for a large proportion of the vegetative cell cycle and exhibited significant resistance to adverse culture conditions. Immunoprecipitation of 35S-labeled soluble Euplotes proteins with PCNA/cyclin autoimmune sera revealed a spectrum of low molecular mass proteins. PCNA/cyclin-like proteins have now been observed in the widely divergent species: human, rat, amphibian, yeast, and ciliated protozoa.

Tomographic reconstruction from energy-filtered images of thick biological sections.

Energy filtration makes it possible to image an approximately 0.5 microm biological section at 80 kV in the electron microscope. Based on spectra taken at different tilt angles, we chose the most probable energy loss, deltaEp +/- 10eV for each tilt angle, as the imaging energy window. A complete tilt series from +60 degrees to -60 degrees at 2.5 degree intervals was collected on the Zeiss EM902 and used in a tomographic reconstruction of transcriptionally active chromatin in the Balbiani ring of Chironomus tentans.

Synthesis of a more stable osmium ammine electron-dense DNA stain.

Specific DNA staining for electron microscopic observation is simplified by a shorter synthesis of the staining reagent. The new, more reliable reagent, osmium ammine-B, is stable for more than a year, dissolves completely in water, and does not require reoptimization of staining conditions for every batch, yet reproducibly gives strong contrast to DNA-containing structures.

Differential distribution of alpha-tubulin isotypes in Euplotes eurystomus determined by confocal immunofluorescence microscopy.

Detergent permeabilized Euplotes eurystomus (a fresh water hypotrichous ciliate) was reacted with monoclonal and polyclonal antibodies specific for either detyrosinated or tyrosinated alpha-tubulin (Glu- or Tyr-tubulin). The isolated cytoskeleton-nuclear complex was examined by Western immunoblotting and by immunofluorescent and electron microscopic methods. Both Glu- and Tyr-tubulins were detected by immunoblot analysis. Immunofluorescent microscopy indicated that the alpha-tubulin isotypes are concentrated in different regions of permeabilized cells: Glu-tubulin is located primarily in cirri, membranelles, and surrounding the macro- and micronuclei. Tyr-tubulin is principally at the bases of cirri and membranelles. This differential distribution of alpha-tubulin isotypes is discussed in terms of current concepts concerning the correlation of tubulin post-translational modifications to microtubule stability. Confocal immunofluorescent imaging was of critical importance in clearly differentiating the Glu-tubulin isotype surrounding the macro- and micronuclei from a brilliantly fluorescent environment originating from cytoskeletal structures. In conjunction with conventional and stereo-electron microscopy, confocal optical microscopy provided convincing evidence for a "basket" of microtubules surrounding both nuclei.

Energy filtered STEM imaging of thick biological sections.

Energy filtered imaging of thick biological specimens was analysed using a dedicated STEM fitted with an energy loss spectrometer and interfaced with a sophisticated data collection setup. All images were digital, thus permitting a quantitative analysis of the data. We also present a mathematical explanation of the data, which is useful in predicting the quality of thick specimen images formed with energy filtered electrons. It is known that increasing specimen thickness leads to a decrease of the zero energy loss intensity and an increase in higher (multiply scattered) energy loss electrons. We show that contrast decreases gradually with increased energy loss but, most important, the signal to noise ratio is maximal at an energy loss position slightly below the intensity maximum. This is the optimal position for imaging thick specimens. Moreover our studies confirm that the following parameters have similar effects on the energy loss spectra: (1) increased thickness (t); (2) higher average Z number elements (or lower mean free path); and (3) lower primary voltage (V0).

Replication bands and nucleoli in the macronucleus of Euplotes eurystomus: an ultrastructural and cytochemical study.

The principal structural compartments of the macronucleus of Euplotes eurystomus were examined by ultrastructural and cytochemical procedures. Interphase chromatin is condensed in highly compact granules that stain intensely with the DNA-specific osmium-amine procedure. Nucleoli react strongly with silver and with thiol-specific reagents, but are almost completely unstained by osmium-amine. The organelle of DNA synthesis, the replication band, is composed of 2 zones. The forward zone consists of highly ordered chromatin fibers, stains strongly with osmium-amine, with silver, and with thiol-specific reagents. The rear zone, which is the site of DNA synthesis, is impoverished in DNA, and is very sensitive to collapse induced by in vivo heat shock, or during nuclear isolation.

Lectin-like components in the macronuclear replication bands of Euplotes eurystomus.

Upon incubation with fluoresceinylated neoglycoproteins, isolated macronuclei from the ciliated protozoan Euplotes eurystomus display different labelling patterns depending on the nature of the sugar bound to the neoglycoproteins. Specific sugar-binding components (i.e., lectin-like molecules) are associated with presumed nucleoli and with the macronuclear replication bands. This is the first demonstration that DNA synthesis and sugar-binding components are co-localized in an eukaryotic cell.

In vitro DNA synthesis in the macronuclear replication band of Euplotes eurystomus.

Isolated macronuclei from the hypotrichous ciliated protozoan Euplotes eurystomus incorporate biotinylated dUTP specifically into the replication band (RB) as detected with immunofluorescence, using rabbit anti-biotin antibodies followed by fluorescein-conjugated goat anti-rabbit IgG. When gold-conjugated goat anti-rabbit IgG was used in a preembedded reaction, subsequent immunoelectron microscopic analysis demonstrated that the biotinylated nucleotide appeared more concentrated in the rear zone of the RB, with almost no labeling in the forward zone. It was possible to use the immunofluorescent assay to establish that incorporation of biotinylated dUTP is inhibited by simultaneous addition of N-ethyl maleimide or aphidicolin, and by omission of any one of the other unlabeled dNTPs. In addition, prolonged heat shock of the intact cells, before lysis and in vitro assay, yielded markedly reduced incorporation. Comparison with published data on the in vivo incorporation of [3H]thymidine into Euplotes eurystomus RBs indicates the fidelity of the in vitro reaction.