Involvement of nuclear lamins in postmitotic reorganization of chromatin as demonstrated by microinjection of lamin antibodies

The nuclear lamins are major components of a proteinaceous polymer that is located at the interface of the nuclear membrane and chromatin; these lamins are solubilized and dispersed throughout the cytoplasm during mitosis. It has been postulated that these proteins, assembled into the lamina, provide an architectural framework for the organization of the cell nucleus. To test this hypothesis we microinjected lamin antibodies into cultured PtK2 cells during mitosis, thereby decreasing the soluble pool of lamins. The antibody injected was identified, together with the lamins, in cytoplasmic aggregates by immunoelectron microscopy. We show that microinjected cells are not able to form normal daughter nuclei, in contrast to cells injected with other immunoglobulins. Although cells injected with lamin antibodies are able to complete cytokinesis, the chromatin of their daughter nuclei remains arrested in a telophase-like configuration, and the telophase-like chromatin remains inactive as judged from its condensed state and by the absence of nucleoli. These results indicate that lamins and the nuclear lamina structure are involved in the functional organization of the interphase chromatin.     

Cytochemical properties of interphase chromatin condensed as a result of treatment with caffeine

Treatment of living cultured cells with caffeine (10 mg/ml, 2 h, 37 °C) brings about marked chromatin condensation which results in the appearance of small, distinct chromatin clumps in the majority of interphase nuclei. The changes taking place in the chromatin properties under the action of caffeine are rather similar to those observed in mitotic condensation (an increase in acridine orange and berberine binding and a sharp decrease in [³H]actinomycin D binding in situ; inhibition of [³H]uridine incorporation in vivo) and are reversible from the point of view of the criteria studied (nuclear morphology, ligand binding, [³H]uridine incorporation, culture viability). It is concluded that caffeine treatment can be regarded as a promising approach to the study of events occurring in chromatin condensation.     

Hepatocyte cell surface polarity as demonstrated by lectin binding

We performed an investigation at the ultrastructural level of the differential distribution of lectin-binding sites among sinusoidal, lateral, and bile canalicular domains of adult rat hepatocytes. Lectin binding to hepatocyte glycocalices was studied in situ or after cellular dissociation by enzymatic (collagenase), chemical (EDTA), and mechanical methods, as well as during cell culture. Using thirteen biotinylated lectins and an avidin-biotin-peroxidase complex (ABC), we have identified lectin-binding sites that are predominantly localized in the bile canalicular [Ricinus communis agglutinin (RCA)] or sinusoidal [Phaseolus vulgaris (PHA)] domains in situ and in mechanically dissociated cells. Lens culinaris (LCA) staining was prominent on sinusoidal surfaces, slight along lateral surfaces, and completely absent in the bile canalicular domain. Concanavalin A (ConA) was unique in binding equally to all domains. Triticum vulgaris [wheat germ agglutinin (WGA)] was also bound to all domains, but most intensely to the bile canalicular region. Cells dissociated via collagenase or EDTA treatment exhibited a spherical morphology characterized by many surface microvilli and absence of morphological domains. Lectin binding to dissociated cells was uniformly distributed over the entire cell surface, suggesting a redistribution of lectin receptors that was independent of the separation procedure. Hepatocytes in culture exhibited a partial restoration of morphological domains, but lectin binding polarity was not re-established.     

Cell surface binding of colloidal iron and Limax flavus agglutinin compared by X-ray microanalysis.

The surface characteristics of tumour cells and T-cell hybridoma variants were examined by quantitative X-ray microanalysis using metal-based probes. Comparisons of populations of cells differing in invasive potential revealed differences in the binding of positive colloidal iron hydroxide, a general probe for anionic sites. Substantial differences between populations were also detected by the binding of Limax flavus agglutinin, which was used in conjunction with fetuin-gold as a specific marker for sialic acid.     

Cytochemistry of colloidal iron binding to the surface of hela cells and human erythrocytes

Summary It seems from the literature that colloidal iron (C.I.) binding sites on cell surfaces cannot be completely removed by treatment with Vibrio Colerae -neuraminidase. We wondered if C.I. particles bind to negative groups other than the carboxyl groups of sialic acids. Using HeLa cells from suspension cultures and fresh human erythrocytes, we examined, with the transmission electronmicroscope, the influence of the following enzymatic and histochemical treatments on C.I. staining: -neuraminidase; hyaluronidase; ribonuclease; -amylase; mild methylation (MM); MM+saponification (Sap.); MM+Sap+MM; MM+Sap+-neuraminidase; active methylation (AM); AM+Sap; AM+Sap+AM; AM+Sap+-neuraminidase; CH₃OH (80%); Sap. It seemed from these experiments that the carboxyl groups of -neuraminidase sensitive sialic acids constitute the majority of binding sites for C.I. to these particular cells. The most interesting candidates for the residual binding of C.I. are carboxyl groups of -neuraminidase resistant molecules, sulfon, sulfin, and sulfate groups.Supported by a grant from the Algemene Spaar- en Lijfrentekas Cancer FundThe authors gratefully acknowledge the technical assistance of L. Baeke, O. Claeys and J. Roels van Kerckvoorde     

Cytochemical study of the elimination chromatin in silkworm meiosis]

The elimination chromatin separating from chromosomes during the first maturation division of female sex cells of lepidoptera insects was studied cytochemically on paraffin sections of eggs of Bombyx mori L. Ocytes at the metaphase-telophase stage of the first meiotic division were stained for RNA by metyl green-pyronin and gallocyanin with negative results. These data differ from earlier positive results for Solenobia triquetrella reported by Ris and Kleinfeld, 1952.     

Measurement of colloidal iron binding at low pH in cartilage using the proton microprobe

Summary Quantitative micro-PIXE analysis was performed on mouse embryo epiphyseal cartilage and on the rib cartilage of mature animals after incubation of sections with colloidal iron at pH 1.8. The iron content as well as that of sulphur and phosphorus and Fe/S, Fe/P ratios were determined. It was found that colloidal iron content was higher in the cartilage than in other tissues. The cartilage also displayed the highest content of sulphur. The Fe/S ratio was however not constant, being highest in the degeneration zone close to the mineralization front, where the binding of iron was strongest while the amount of sulphur decreased. This indicates that factors other than number of sulphate groups influence the binding of positively charged molecules to glycosaminoglycans. This is confirmed by differences in the results obtained for embryonic and mature rib cartilage.     

Troponin bound calcium in electron micrographs of myocardial cells as demonstrated by the potassium-antimonate technique

Summary Following the K-antimonate reaction in atrial myocardial tissue, a pattern of evenly spaced cross striations of antimonate precipitates is demonstrated along the myofilaments. This spacing, found in both turtle and mouse atria, has a periodicity of about 400 Å. In order to test the shifts of the antimonate reaction product in the tissue, a comparison is made between the localization of the antimonate precipitate as seen in viz. thin plastic sections and in cryo-ultra sections being dry-cut at -90° C from N₂ frozen tissue. Preliminary results suggest only minor distributional differences in the sarcomeric pattern. On the basis of these tests, and, on the basis of previous studies by means of X-ray microanalysis, it is suggested that the periodic pattern of evenly spaced precipitates, reflects the localization of troponin bound calcium along the thin filaments during contraction.This work was supported by grants from The Norwegian Research Council for Science and the Humanities. We are also indebted to Mrs. Trine Jensen and Miss Sigrid Devik for skilful technical assistance.     

Attachment of human chromatin fibers to the nuclear membrane,as seen by electron microscopy

Summary Whole-mount preparations and thin sections of human interphase cells and metaphase chromosomes were examined by electron microscopy. Irregularly folded, 250 Å thick fibers, which is the basic substructure of inactive chromatin and mitotic chromosomes, were found to be firmly attached to the annuli of the inner nuclear membrane. At metaphase, fragments of the nuclear membrane were seen to adhere to the chromatids. Single fibers stretching out from the telomeres were observed connecting chromatids of nonhomologous chromosomes. A possible model of DNA replication at the nuclear pore complex is presented.

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Zusammenfassung Totalpräparate und Dünnschnitte menschlicher Interphase-Zellen und Metaphase-Chromosomen wurden mit dem Elektronenmikroskop untersucht. Unregelmäßig gefaltete, 250 Å dicke Fäden bilden die Grundstruktur des inaktiven Chromatins und der Mitose-Chromosomen. Diese Fäden hängen in der Interphase an vielen Stellen fest an der inneren Kernmembran an den annuli der Kernporen. In der Metaphase sind häufig noch Reste der Kernmembran durch Fäden mit den Chromatiden verbunden. Einzelne, jeweill vom Telomer ausgehende Fäden verknüpfen Chromatide nichthomologer Chromosomen. Das Modell einer möglichen DNA-Replikation an den Poren der Kernmembran wird diskutiert.

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Supported by a grant (La 185/3) of the Deutsche Forschungsgemeinschaft.     

Decrease in human lymphocyte surface glycoconjugates in leukemia as demonstrated by lectin binding

Qualitative variations in the glycoconjugates which make up the lectin receptor sites on the membranes of leukemic lymphocytes, compared with those of normal cells, have been studied by the use of three tritiated lectins: Robinia pseudoacacia lectin, Concanavalin A and Ricinus communis (var. Sanquineus) agglutinin (RCA 120). The binding specificity of these lectins has been demonstrated using specific determinants: alpha-methylmannoside and galactose for Concanavalin A and Ricinus communis agglutinin respectively. For the Robinia lectin this specificity was determined by saturation of the receptor sites with the unlabeled Robinia lectin before the addition of isotopically labeled Robinia lectin. The results show a decrease in the number of receptor sites on the leukemia cells, especially in chronic lymphoid leukemia, relative to that on normal cells. The apparent affinity constants of leukemic cells in all cases remain higher than those of normal cells.     

Pitfalls in ecto-5''-nucleotidase enzyme cytochemistry as demonstrated by the immunogold-labelling technique on macrophages

Summary We have used both the enzyme cytochemical method with lead nitrate as a capture agent and an immunological method at the electron microscope level to localize plasma membrane 5-nucleotidase in rat peritoneal resident macrophages during the initial interactions of latex beads or heat-killedEscherichia coli with the cell during phagocytosis. In macrophages at rest, cytochemical reaction product was evenly distributed along the external surface of the plasma membrane. However, when the cells were phagocytosing latex beads or bacteria, reaction product covered the entire surface of the adhering particles. To determine whether the apparent redistribution of 5-nucleotidase onto the adhering particle was fact or artifact, we localized 5-nucleotidase using a monoclonal antibody and an immunogold labelling technique. In macrophages binding or beginning to ingest bacteria, gold particles were distributed along the plasma membrane, except at the sites of cell-bacterium internalization. More significantly, the adhering bacteria were free of gold particles and therefore had no 5-nucleotidase on their surfaces. Latex beads proved to be unsuitable as a test particle because the gold particles stuck to them non-specifically. We conclude that the artifactual redistribution of lead-phosphate reaction product is a major drawback of enzyme cytochemical methods when used on cell surfaces and that the immunogold labelling technique is more reliable.     

Cytochemistry of colloidal iron binding to the surface of Hela cells and human erythrocytes.

It seems from the literature that colloidal iron (C.I.) binding sites on cell surfaces cannot be completely removed by treatment with Vibrio Colerae alpha-neuraminidase. We wondered if C.I. particles bind to negative groups other than the carboxyl groups of sialic acids. Using HeLa cells from suspension cultures and fresh human erythrocytes, we examined, with the transmission electronmicroscope, the influence of the following enzymatic and histochemical treatments on C.I. staining: alpha-neuraminidase; hyaluronidase; ribonuclease; alpha-amylase; mild methylation (MM); MM + saponification (Sap.); MM + Sap +MM; MM + Sap + alpha-neuraminidase; active methylation (AM); AM + Sap; AM + Sap + AM; AM + Sap + alpha-neuraminiadase; CH3OH (80%); Sap. It seemed from these experiments that the carboxyl groups of alpha-neuraminidase sensitive sialic acids constitute the majority of binding sites for C.I. to these particular cells. The most interesting candidates for the residual binding of C.I. are carboxyl groups of alpha-neuraminidase resistant molecules, sulfon, sulfin, and sulfate groups.     

Sodium binding in Halobacterium halobium measured by the nuclear magnetic resonance technique

Relaxation time measurements T₁ and T₂ of sodium in Halobacterium halobium pellets were carried out at two frequencies. From those measurements, combined with intensity measurements of the sodium in the system, estimation of the properties of the sodium ions in the system was carried out. It is suggested that three types of sodium ions are present in the bacterial pellet. (A) The extracellular sodium with properties of free solution and (B) sodium which is in the pericellular volume between the cell wall and the cell membrane. There is an exchange between type A and type B sodium. The type B sodium has (e²qQ)/h = 3.7 · 10⁷ rad/s, τ_cB = 5.2 · 10⁻⁶ s and τ_B = 1 · 10⁻³ s. The sodium of type C is bound inside the cell and undetected. It's concentration inside the cell is assumed to be 1.9 M.     

Sugar binding properties of the vitellogenic proteins in the Colorado beetle,demonstrated by hemagglutination and precipitation experiments

Summary The sugar binding properties of 2 important vitellogenic proteins in Colorado beetle hemolymph were demonstrated by hemagglutination and precipitation experiments. The agglutination of human red blood cells by the hemolymph of reproducing females was observed up to a hemolymph dilution of 1/256, irrespective of the blood-group. It increased significantly after trypsinization of the crythrocytes. Vitellogenin 1 was identified as the hemagglutinin. Hemagglutination and hemagglutination inhibition tests showed that this protein has a low affinity for hexosamines and a higher affinity for sulfated polysaccharides. Precipitation tests demonstrated that besides vitellogenin, another major yolk protein, chromoprotein 2, reacts with sulfated polysaccharides. The possibility that there is a specific reaction of the vitellogenic proteins with well defined saccharides on the oocyte surface is discussed. This lectintype reaction may explain the selectivity of yolk precursor endocytosis.