Characterization of cation-rich follicle cells in vitellogenic follicles of Drosophila melanogaster

Abstract. Cations in the ovarian follicles of Drosophila were precipitated with potassium antimonate and the distribution of the precipitates in the follicle cells was studied. In three stage 10 follicles, the precipitate density in the different follicle cells was determined by counting the number of precipitates per unit area in an electron microscope. Among the follicle cells, those migrating centripetally between the nurse cells and oocyte were found to differ widely in their precipitate density. When stage 10B follicles at different substages were compared, the number of precipitaterich follicle cells was found to increase with developmental age. At middle and late stage 10B, groups of cation-rich follicle cells extended from the region of the ring canals to the outer face of the follicle. We also found changes in the precipitate density of the border cells during vitellogenesis. The precipitate-rich follicle cells may play a role in the postulated electrical current flow.     

Doubling of cell mass is not necessary in order to achieve cell division in cultured human cells

When exponentially growing NHIK 3025 cells were shifted from medium containing 30% serum to medium containing 0.03% serum the rate of net protein accumulation was reduced due to both a reduction in the rate of protein synthesis and an increase in the rate of protein degradation. This change in growth conditions increased the protein doubling time from 18 to 140 h. The cell cycle duration of cells synchronized by mitotic selection was, however, only increased from 17 to 26 h by this treatment. Therefore, when the cells divide by the end of the first cell cycle following synchronization, the cells shifted to 0.03% serum contained far less protein than those growing continuously in 30% serum. Hence, the attainment of a critical cell mass is probably not controlling cell division for cells growing in a balanced state.     

Identification of a nuclear and of a cytoplasmic polypeptide whose relative proportions are sensitive to changes in the rate of cell proliferation

A 36K MW polypeptide was previously identified by two-dimensional gel electrophoresis whose relative proportion increases in S phase of HeLa cells, and following viral transformation of mouse 3T3B and hamster BHK21 cells. We report here experiments that show that this polypeptide (isoelectric focusing (IEF 49)) is localized in the nucleus and that its relative proportion decreases dramatically under conditions for which there is a decrease in the rate of cell proliferation, due for example, to the formation of giant HeLa cells by X-ray irradiation or the senescence of human skin fibroblasts. It is present in negligible amounts in non-cycling cells of adult tissues. These studies also revealed and unrelated cytoplasmic polypeptide (IEF 52; tropomyosin related; coordinates 35/1.43) whose relative proportion increased significantly and reproducibly with a decrease in the rate of cell proliferation. It is suggested that IEF 49 may be a useful marker for cycling cells.     

Induction of differentiation in a human promyelocytic leukemic cell line (HL-60). Production of granule proteins

Serum fibronectin inhibits the adhesion of neutrophil granulocytes (PMNs) to clean glass, HSA-coated glass, and gelatin-coated glass. It does not affect adhesion to collagen-coated glass which itself provides a substratum of low adhesiveness for PMNs. Cell-cell adhesion is not affected. During the acute inflammatory response in vivo, PMNs must migrate through the fibronectin and collagen containing extracellular matrix: reducing cell-substratum adhesion in these circumstances might facilitate locomotion towards inflammatory foci.     

Immunocytochemical localization of chromatin regions UV-microirradiated in S phase or anaphase. Evidence for a territorial organization of chromosomes during cell cycle of cultured Chinese hamster cells

Chinese hamster cells (M3-1 line) in S phase were laser-UV-microirradiated (lambda, 257 nm) at a small site of the nucleus. Cells were fixed either immediately thereafter or in subsequent stages of the cell cycle, including prophase and metaphase. The microirradiated chromatin was visualized by indirect immunofluorescence microscopy using antibodies specific for UV-irradiated DNA. During the whole post-incubation period (4-15 h) immunofluorescent labelling was restricted to a small part of the nucleus (means, 4.5% of the total nuclear area). In mitotic cells segments of a few chromosomes only were labelled. Following microirradiation of chromosome segments in anaphase, immunofluorescent labelling was observed over a small part of the resulting interphase nucleus. A territorial organization of interphase chromosomes, i.e. interphase chromosomes occupying distinct domains, has previously been demonstrated by our group for the nucleus of Chinese hamster cells in G1. Our present findings provide evidence that this organization pattern is maintained during the entire cell cycle.     

The distribution of tau and HMW microtubule-associated proteins in different cell types

To investigate the distribution of the tau and HMW microtubule-associated proteins (MAPS) and their relationship to microtubules in vivo, we have examined a wide variety of avian and mammalian cell types by immunofluorescence with antisera to these two proteins. Anti-HMW serum stains cytoplasmic microtubules in all mammalian cell types so far examined. However, anti-tau serum did not stain cytoplasmic microtubules in rat glial cells or in pig kidney cells. In mammalian neurons, fibroblasts and neuroblastoma cells, the staining of microtubules with both sera was similar. Anti-HMW serum did not stain primary cilia or cilia on isolated tracheal epithelial cells, whereas anti-tau serum did stain these ciliary microtubules. We believe these results indicate that some types of microtubules may be associated with only the tau or the HMW protein, whereas others may be associated with both tau and HMW protein. With respect to avian cells, anti-HMW serum did not stain microtubules in any of the three cell types examined, whereas the anti-tau serum stained them in two cell types. Furthermore, double diffusion tests indicated that anti-pig tau serum will precipitate both pig brain tau and tau protein isolated from chick brain, whereas anti-HMW serum will precipitate only pig brain and not chick brain HMW protein. We believe tau protein is antigenically similar in both avian and mammalian cells, whereas the HMW protein from these two sources is antigenically distinct.     

Modification of the surface ATPase activity in cultured hepatoma cells by lipid-depleted media

Several compounds have been described which elute fibronectin from a gelatin-Sepharose affinity support. In the present study, it has been found that the potent chaotrophic agent, lithium di-iodosalicylic acid, is 20-fold more effective in eluting fibronectin from collagen than any other presently described fibronectin elution agent. Lithium di-iodosalicylic acid and certain other fibronectin elution agents have been characterized in regard to several parameters involved in the elution of fibronectin from collagen and plastic substrata. By assaying for retention of the cell adhesive activity of fibronectin, it has been demonstrated that 8 M urea + 0.1 M citric acid, pH 4.7, is the most effective condition for preservation of biological activity following elution of fibronectin from the gelatin-Sepharose affinity support.     

Regulation of synthesis of the larval serum proteins of Drosophila melanogaster

We have determined the relative amounts of subunits of larval serum proteins (LSPs) 1 and 2 during larval development in Drosophila melanogaster. These results indicate that synthesis of polypeptide subunits of LSP-1 and LSP-2 is coordinate: the proteins are first detected at the same time; they accumulate in a coordinate fashion; their RNAs are first detected at the same time; the RNAs also accumulate in similar relative amounts. Analyses of fat body polypeptides and fat body RNA indicate that synthesis of LSP-1 declines at a time when there are still substantial quantities of LSP-1 RNA in the cytoplasm. Cessation of LSP-1 subunit synthesis occurs before cessation of LSP-2 synthesis, indicating that at late times the genes (or mRNAs) for these two proteins are subject to different "switch-off" controls.     

Occurrence of two different intermediate filament proteins in the same filament in situ within a human glioma cell line : An immunoelectron microscopical study

Intermediate filament systems of an established glioma cell line have been characterized by double immunofluorescence microscopy and by immunoelectron microscopy using two antibodies, one of which recognizes glial fibrillary acid protein (GFA) but not vimentin, and the second which recognizes vimentin but not GFA. The results show that glioma cells express two immunologically distinct IF polypeptides which are found in the same 10-nm filaments. Juxtanuclear caps formed after exposure of the cells to colcemid consisted of intermediate filaments composed of both GFA and vimentin. In immunoelectron microscopy both untreated cells and cells treated with colcemid show discontinuous labelling when only a single antibody is used, but continuous labelling when both antibodies are used simultaneously.     

Multinucleate plant cells : II. Requirements for nuclear DNA synthesis

A synchronous population of multinucleate cells has been experimentally induced in Allium cepa root meristems by a chemical method that involves the induction of aneuploidy in individual nuclei. Whereas all multinucleate cells as a whole present a tetraploid chromosome complement, their aneuploid nuclei constitute compartments containing only part of the parental genetic material. We analysed the degree of synchrony in the course of S period progression in the unbalanced nuclei sharing a common cytoplasm (bi-, tri- and tetranucleate cells). In most of the multinucleate cells the nuclei entered DNA replication simultaneously, but a significant proportion of cells showed labelled and unlabelled nuclei which was due to the absence of DNA synthesis in some aneuploid nuclei. These nuclei lacked replicative capacity, but did not inhibit DNA synthesis of the replicating nuclei. Our findings confirm the existence of cytoplasmic factor(s) inducing the synchronous initiation of S period, and suggest strongly the occurrence of intranuclear factor(s)—likely gene products—as a requirement for the onset of DNA synthesis itself in every nucleus.     

Cartilage-derived factor (CDF) : II. Somatomedin-like action on cultured chondrocytes

Previously, we showed that fetal bovine cartilage contains a polypeptide that stimulates the incorporation of [³⁵S]sulfate into proteoglycans synthesized by rat and rabbit costal chondrocytes in culture. In this paper, we report that the cartilage-derived factor (CDF) increases not only [³⁵S]sulfate incorporation but also [³H]thymidine incorporation into rabbit chondrocytes in monolayer culture. The dose-response curve of CDF stimulation of DNA synthesis was similar in profile to that of CDF stimulation of proteoglycan synthesis. In addition, CDF markedly enhanced [³H]uridine incorporation into rabbit chondrocytes and significantly enhanced [³H]serine incorporation into total protein. These findings indicate that fetal bovine cartilage contains a factor that shows somatomedin-like activity in monolayer cultures of rabbit chondrocytes.     

The tumor promoter, 12-O-tetradecanoylphorbol-13-acetate accelerates keratinocyte differentiation and stimulates growth of an unidentified cell type in cultured human epidermis

Studies were conducted to determine the effects of the mouse skin tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) on cultured human epidermal cells for comparison with known effects on mouse keratinocytes. In contrast to its effect on mouse cells, TPA did not stimulate human epidermal cell DNA synthesis. TPA stimulated differentiation in human keratinocytes resulting in sloughing of many cells by the 3rd day after exposure. Quantitative assays revealed that 50% of the TPA-exposed population was composed of cornified cells as opposed to 8% in untreated controls. A morphologically distinct cell type (TT cell) emerged after TPA treatment which was triangular in shape, did not stratify, appeared to proliferate rapidly and at most TPA concentrations became the predominant cell type within 1–2 weeks. Cultures composed predominantly of TT cells formed few cornified envelopes, grew well in the absence of TPA and formed colonies at low cell input. In contrast to its effect on keratinocytes, TPA enhanced TT colony formation 3–4-fold and decreased the doubling time of TT cells. Studies were performed to determine the origin of TT cells. Immunofluorescent staining indicated that TT cells lacked the keratinocyte antigens keratin, pemphigus and pemphigoid. Tonofilaments and desmosomes were not seen by electron microscopy. The lack of both melanosomes and standard histochemical DOPA oxidase staining indicated that TT cells were probably not of melanocyte origin. Tests used to identify Langerhans cells were negative. Whereas TT cells, as well as dermal fibroblasts, yielded positive immunofluorescence with antibodies to vimentin, TT cells gave a weak histochemical leucine aminopeptidase reaction, while the reaction of fibroblasts exposed to TPA was strong. Treatment of human dermal fibroblasts with TPA did not yield TT cells. The endothelial cell antigen factor VIII-associated protein was absent by immunofluorescence. These results suggest that the primary effect of TPA on cultured human epidermis is to accelerate terminal differentiation in the keratinocyte population and to stimulate growth of an as yet unidentified cell type.     

Embryonal carcinoma cell growth and differentiation. Production of and response to molecules with transforming growth factor activity

Transforming growth factors are known to induce anchorage-independent growth of non-transformed cells, and are released by a variety of cells, including MSV-transformed cells. This study demonstrates that the differentiated cells derived from F9 and PC-13 embryonal carcinoma cells, but not the parental cells themselves, respond by increased growth to several factors released by MSV-transformed cells, including partially purified sarcoma growth factor. The chemical properties of the growth-promoting activity are shown to match the chemical properties of the transforming growth factors released by MSV-transformed cells. Furthermore, F9 and PC-13 embryonal carcinoma cells, which do not respond to factors released by MSV-transformed cells, are shown to release factors with transforming growth factor activity. Based on the close relationship between mouse embryonal carcinoma cells and cells of early mouse embryos, it is suggested that molecules with transforming growth factor activity may play a role during the early stages of mammalian development.     

In vivo identification of Tetrahymena actin probed by DMSO induction of nuclear bundles

We report the first successful identification of actin, an ubiquitous contractile protein, in Tetrahymena pyriformis (strain W). We employed dimethyl sulfoxide (DMSO) as a probe to induce the formation of actin bundles in the cell nucleus [1, 2] through disruption of cytoplasmic microfilament organization [3, 4]. The cells were incubated for 30 min at 22 °C in the inorganic medium of Prescott & James [5] containing 10% DMSO, and observed under a transmission electron microscope (TEM). Microfilarment bundles were formed in interphase macronuclei, and these microfilaments, approx. 6 nm in diameter, could be decorated by rabbit skeletal muscle heavy meromyosin (HMM) in the glycerinated model. In many cases, the bundles formed closely parallel to natively existing bundles of microtubules. Interestingly, these microtubules had prominent striation with 15–16 nm periodicity. SDS-polyacrylamide gel electrophoresis was designed to show the low actin content of Tetrahymena cells in comparison with that of Dictyostelium. Actin was suggested to comprise less than 1.7% of the total protein in Tetrahymena, whereas as much as 6% was actin in Dictyostelium cells. In assessing the physiological significance of the bundle formation, we further performed HMM and myosin subfragment-1 (S1)-binding studies to clarify the organization process and the polarity of the DMSO-induced nuclear actin filaments by using the tannic acid staining technique [6]. Randomly oriented short filaments appeared in the nucleus treated with 10% DMSO for 10 min. These filaments became elongated and associated with each other to form loose bundles in the following 10 min. With 30-min treatment, the filaments were organized and large bundles with single axes developed. With these well-developed bundles, the Student's t-test was performed on 172 pairs of neighboring filaments and the probability (p) of the deviation from random polarity was 0.08, suggesting that the filaments were organized in an anti-parallel manner. The results show that the DMSO induction of nuclear actin is a powerful tool to demonstrate the existence of cellular actin in vivo and to study the mechanism of microfilament organization in relation to cell physiological activities.     

Characterization of a detergent-resistant surface lamina in cultured human fibroblasts

Treatment of cultured human fibroblasts with 0.5% Triton X-100 produces substratum-anchored cytoskeletal preparations consisting of cytoplasmic filaments, nucleus and a plasma membrane-derived surface lamina. The lamina was visualized in fluorescence microscopy with fluorochrome-coupled wheat germ agglutinin (WGA) as a lace-like structure, extending throughout the cell domain. It displayed a different organization at the ventral and dorsal surfaces of the cell, partially coaligning with bundles of actin and myosin filaments at the dorsal cell surface. At the ventral surface vinculin patches appeared to be included in the surface lamina. Polyacrylamide gel electrophoresis, combined with lectin reactivity studies and lectin affinity chromatography, revealed a 140 kD sialoglycoprotein as the major glycoprotein component of the surface lamina.     

Considerations in the isolation of rat liver nuclear matrix,nuclear envelope,and pore complex lamina

A number of recent studies have demonstrated a salt-, nuclease, and detergent-resistant subnuclear structure termed the nuclear protein matrix which consists of a fibrogranular intranuclear network, residual components of the nucleolus, and a peripheral lamina. Other workers, however, have shown that somewhat similar methods result in the isolation of the peripheral lamina devoid of the intranuclear components. In this report we demonstrate that seemingly slight changes in the isolation procedure cause major changes in the morphology of the residual structures obtained. When freshly purified rat liver nuclei were digested with DNase I and RNase A and then extracted with buffers of low magnesium ion concentration (LS buffer) and high ionic strength (HS buffer), the resulting structures isolated prior to or after Triton X-100 extraction lacked the extensive intranuclear network and the easily identifiable residual nucleoli present in the nuclear protein matrix. Systematic modification of this extraction procedure revealed that morphologically identifiable residual nucleoli were present when digestion with RNase A followed extraction with HS buffer but were absent when the order of these steps was reversed. The removal of the nucleolus by RNase A and HS buffer correlated with the removal of nuclear RNA by the same treatments. These coordinate events could not be prevented by treatment with protease inhibitors but were prevented by treatment of the RNase A with diethylpyrocarbonate, an RNase inhibitor. The extensive intranuclear network seen in the nuclear protein matrix was sparse or absent when residual structures were prepared from DNase- and RNase-treated nuclei under conditions which minimized the oxidation of protein sulfhydryl groups. In contrast, an extensive non-chromatin intranuclear network was seen if the formation of intermolecular protein disulfide bonds was promoted by extraction of nuclei with cationic detergents, by overnight incubation, or by treatment with oxidizing agents like sodium tetrathionate prior to nuclease digestion and subsequent extraction. By varying the order of extraction steps and the extent of disulfide cross-linking, it is possible to isolate from a single batch of nuclei residual structures with a wide range of morphologies and compositions.     

Tetrahymena calmodulin. Characterization of an anti-tetrahymena calmodulin and the immunofluorescent localization in Tetrahymena

A rabbit antiserum specific for Tetrahymena calmodulin was prepared and characterized: In Ouchterlony's immunodiffusion test, the antiserum gave rise to a single precipitin line only with calmodulin in the reaction with crude Tetrahymena extract and the antiserum cross-reacted with a calmodulin fraction from Paramecium, but not with several calmodulin fractions, from higher organisms. Calmodulins from the ciliates appear to share some antigenic determinants which are absent in calmodulins from higher organisms. The intracellular localization of calmodulin was investigated by indirect immunofluorescent method using anti-Tetrahymena calmodulin antibody purified on an antigen-Sepharose affinity column. Immunofluorescence was localized in the oral apparatus, cilia, basal bodies, the anterior end of the cell, and the contractile vacuole pores. The localization suggested involvement of calmodulin in food vacuole formation (nutrient uptake), excretion of contractile vacuole contents (regulation of osmotic pressure), and in ciliary movement (reversal). The suggestion was supported by the observation that trifluoperazine markedly suppressed food vacuole formation and excretion of contractile vacuole contents and affected the ciliary motion.     

Isolation of cloned variants of a rat hepatoma cell line with altered attachment to collagen, but normal attachment to fibronectin

Cloned variants of a rat hepatoma cell line have been isolated which exhibit normal attachment and spreading behavior on fibronectin substrata, but which are defective in their ability to attach to native collagen films. These clones should be useful for identifying specific macromolecules involved in the cell-to-collagen interaction.     

Evidence for the association of two cell surface glycoproteins of 13762 mammary ascites tumor cells : Concanavalin A-induced redistribution of peanut agglutinin-binding proteins

The behavior of the cell surface concanavalin A (conA) receptors and of peanut agglutinin (PNA) receptors on the MAT-B1 ascites subline of the 13762 rat mammary adenocarcinoma was examined using fluorescein-labeled conA and PNA. ASGP-1, the major glucosamine-containing glycoprotein of these ascites cells, is the only PNA-binding protein observed by dodecyl sulfate electrophoresis. ASGP-2, the second most prominent component after glucosamine labeling, is the most abundant conA-binding protein. These two glycoproteins were previously shown to be associated as a complex in detergent extracts of the cells [20]. ConA-binding proteins, upon incubation with fluorescein-labeled conA (FITC-conA), redistribute on the cell surface into small and large aggregates similar, but not identical, to those seen in ‘patching’ and ‘capping’ experiments with lymphocytes. PNA-binding proteins failed to redistribute during incubation with fluorescein-labeled PNA (FITC-PNA) and appeared in a diffusely stained pattern around the circumference of the cells. However, when cells were treated with unlabeled conA followed by FITC-PNA, or with FITC-PNA followed by unlabeled conA, there was marked redistribution of the FITC-PNA. These results indicate that ASGP-1 redistributes in response to the movement of conAbinding proteins and supports our hypothesis that ASGP-1 and ASGP-2 are associated on the plasma membrane at the cell surface as well as in detergent extracts.     

The culture of chick embryo chondrocytes and the control of their differentiated functions in vitro. I. Characterization of the chondrocyte-specific phenotypes

We have maintained chick embryo chondrocytes in culture for more than 2 months, passaging the floating cells in the absence of ascorbic acid. Throughout the culture period some of the cells attached to the dish, assuming an epithelial-like morphology and subsequently giving rise to new floating cells. The interconversion of the two cell populations was highest in primaries and decreased with the aging of the culture. Cartilage cells synthesized pro-alpha 1 (II) collagen and sulphated proteoglycans in vitro; compared with floaters, the epithelial-like cells secreted relatively large amounts of fibronectin. When ascorbic acid was added to the medium, all cells attached, maintaining their rounded shape; in this condition the pro-alpha, (II) collagen was matured and collagen fibres were detectable outside the cells. Other specific proteins synthesized by the chondrocytes in culture were also identified. One of these, a 64 K collagenase-sensitive protein, was not related to the type II collagen and may represent a new collagen type.