Role of the Cell Surface of Methanosarcina mazei in Cell Aggregation

Colonial aggregates of Methanosarcina (= Methanococcus) mazei were examined with scanning and transmission electron microscopy. Cells are irregular and grouped into multicellular sarcinal colonies, which may disaggregate in older cultures. The protoplast is bounded by a typical trilaminar plasma membrane, outside of which is a matrix of loose fibrils. The presence and compactness of matrix material are responsible for the close packing of cells, and colony disaggregation seems to be the result of matrix shedding and degradation. The cell envelope contains complex hetero polysaccharides of N-acetylgalactosamine and galacturonic and glucuronic acids. Polymers extruded by M. mazei are likely quite adhesive in nature, accounting for its strong adherence to surfaces and hardiness compared with many other methanogens.     

Formation of the plasma membrane during regeneration ofParamecium caudatum

Summary Fragments ofParamecium caudatum cells obtained by merotomy were fixed in 1% OsO₄ within 5 seconds after cutting. The ultrastructure of the damaged area of the fragment was studied in oriented ultrathin sections and by scanning electron microscopy. The cytoplasm exposed by merotomy was covered during a few seconds with a new membrane. This was a typical trilaminar membrane continuous with the plasma membrane covering the undamaged surface of the cell. The surface over the wound was wrinkled into irregular grooves and ridges. The cytoplasm, mitochondria and trichocysts in the injured region were electron translucent. The cytoplasm under the new membrane contained an unusually high amount of small membrane vesicles, 20–90 nm in diameter. These were probably the remnants of subpellicular alveoli and the plasma membrane destroyed by microsectioning. The possibility that the exposed cytoplasm would be covered by mere shifting of the existing plasma membrane can be excluded. The complex structure of the cortex with its subpellicular alveoli and regularly distributed cilia provide a strong argument against this notion. It seems probable that the new membrane was built up from the available molecular material,e.g., phospholipids and proteins present in the cytoplasm. Fragments of the membrane and alveolar membranes in the form of small vesicles may have also been included into the new membrane.     

Outer membrane protein OmpF involved in the transportation of polypyridyl ruthenium complexes into Escherichia coli

The discovery that OmpF was related to the transportation of ruthenium complexes through cell membrane was achieved with proteomics technologies. An integral ruthenium complex exists inside the cell as identified by matrix-assisted laser desorption ionization (MALDI) mass spectrometry. An inhibition assay with Escherichia coli was used to demonstrate the relationship between the transportation of the polypyridyl ruthenium complexes and the presence of OmpF (outer membrane protein F). For instance, the amount of [Ru(tpy)(bpy)Cl]⁺ (tpy: teripyridine; bpy: bipyridine) that entered the cells was determined by inductively coupled plasma optical emission spectroscopy (ICP-OES) of cell extracts and was measured to be approximately 0.55 μM. In the presence of 10% sucrose solution which is known to reduce the OmpF concentration, the ruthenium complex concentration was reduced to approximately 0.28 μM, which is a 50% reduction. These data suggest that OmpF plays a key role in the transportation of positively charged polypyridyl chlororuthenium complexes into E. coli.     

Indirect immunofluorescence localization of ponticulin in motile cells

Ponticulin is the major actin-binding integral glycoprotein in plasma membranes isolated from log-phase Dictyostelium discoideum amebae. As such, this protein appears to be an important link between the plasma membrane and actin filaments (Wuestehube and Luna: Journal of Cell Biology 105:1741-1751, 1987). In this study, indirect immunofluorescence microscopy was used to examine the distribution of ponticulin in randomly moving D. discoideum amebae and in amebae engaged in cell migration and phagocytosis. Ponticulin is distributed throughout the plasma membrane and also is present in intracellular vesicles associated with the microtubule-organizing center-Golgi complex adjacent to the nucleus. In aggregating amebae, ponticulin is concentrated in regions of lateral cell-cell contact and in arched regions of the plasma membrane. Ponticulin also is present, but not obviously enriched, in filopodia, in the actin-rich anterior end of polarized cells, and in detergent-insoluble cytoskeletons. In amebae engaged in phagocytosis of yeast, ponticulin is present but not enriched in phagocytic cups and is associated with intracellular vesicles around engulfed yeast. These results suggest that ponticulin is stably associated with actin filaments in certain regions of the plasma membrane and that the actin-binding activity of ponticulin may be tightly controlled. Indirect immunofluorescence microscopy and immunoblot analysis demonstrate that human polymorphonuclear leukocytes also contain a 17 kD protein that specifically cross-reacts with antibodies affinity-purified against D. discoideum ponticulin. As in D. discoideum, the mammalian 17 kD ponticulin-analog appears to be localized in plasma membrane and is evident in actin-rich cell extensions. These results indicate that ponticulin-mediated linkages between the plasma membrane and actin may be present in higher eukaryotic cells.     

Histochemical staining of the complex carbohydrates of the midgut of the mosquito,Culex tarsalis coquillett

Histochemical staining of the midgut epithelial cell surface complex carbohydrates of the mosquito Culex tarsalis was examined electron microscopically. The microvillar surface is composed primarily of neutral vic-glycoconjugates; positively stained by silver methenamine and silver protein. Lanthanum and alcian blue staining indicate that the microvilli contain a minimal anionic component; possibly phosphoglycoconjugates. Similarly, the intercellular junctions contain a predominance of neutral vic-glycoconjugates. In addition, the intercellular junctions contain fixed positive charges, based on en bloc phosphotungstic acid staining. The midgut basolateral membrane system and the basal lamina are both highly anionic; stained by ruthenium red, tannic acid, alcian blue and periodic acid-chromic acid-phosphotungstic acid. The basolateral plasma membrane also contains some vic-glycoconjugates. Selective staining indicates that the anionic component of the basolateral plasma membrane and the basal lamina is predominantly carboxyl groups; no specific staining for sulfo- or phosphoglycoconjugates was observed.     

Corpuscular and trilaminar appearance of cultivated chick embryo fibroblast plasma membranes

Summary Electron microscope examination of chick embryo fibroblasts grown in tissue culture and fixed in a variety of osmium fixatives as well as in fixatives not containing osmium revealed a limiting cell membrane with a corpuscular substructure. A trilaminar appearance was more difficult to demonstrate in plasma membranes of cells fixed in osmium or glutaraldehyde solutions followed by osmication, while many extensive regions of membranes with this appearance were observed after fixation in buffered potassium permanganate.A study of the conditions favorable to the demonstration of a trilaminar appearance indicated that the level of focus of the electron microscope image and alteration of the intrinsic structure of the summits within the membrane played significant roles in producing this appearance.This work was aided by Research Grant GB 2129 from the National Science Foundation. Some of the equipment used was purchased with funds from the National Institutes of Health Grant 2 TI GM 326. I wish to thank Dr. Robert M. Dougherty from the Department of Microbiology who grew and supplied me with the chick embryo fibroblast cultures used in these studies, and Mrs. Ursula Feller and Miss Leslie Hammack for their technical assistance.     

Calcium,cellular adhesion and aggregation competence in the cellular slime mold Polysphondylium violaceum

Cellular adhesion in Polysphondylium violaceum is mediated by Ca²⁺ ions. The extent of cell adhesion exhibited by developing P. violaceum is greater in the presence of 0.5 mM Ca²⁺ than in the absence of Ca²⁺. Vegetative amebae exhibit some adhesive properties, although the cellular interactions expressed by vegetative amebae are not as extensive as those exhibited by developing amebae. If the amebae are incubated in the presence of chelators (EGTA or EDTA) cellular adhesion is prevented and the amebae remain as single cells. Vegetative cell adhesion is blocked by 1 mM EGTA, whereas blocking adhesion in developing cells requires 5- to 10-fold greater concentrations of EGTA. The acquisition of developmental adhesive properties occurs even if the amebae are incubated in the presence of EGTA, suggesting that Ca²⁺ is required for interaction between adhesion sites but not for their formation. P. violaceum amebae become aggregationcompetent (aggregate immediately when placed on a solid surface) at the same time that the developmental adhesion sites are expressed, even when incubated in the presence of EGTA. Thus it seems unlikely that cellular adhesion is required to develop aggregation competence.     

NUCLEOCYTOPLASMIC EXCHANGES DURING EARLY INTERPHASE

Colloidal gold was injected into the cytoplasm of amebae (Amoeba proteus) approximately 5 min, 1 hr, and 2 hr after cytokinesis. Later, interphase cells were similarly treated. All of the amebae were fixed about 50 min after injection and were examined with the electron microscope in order to determine the distribution of the gold. It was found that for a period of 2 hr after division the uptake of gold by the nuclei was significantly greater than that during late interphase. Correlation of the gold distribution with the morphology of the nuclear envelope indicated that an inverse relationship exists between the rate of incorporation of colloid into the nucleoplasm and the degree of reconstitution of the fibrous lamina ("honeycomb" structure). These data support the view that the fibrous lamina functions in regulating nucleocytoplasmic exchanges.     

Cortical dynein is critical for proper spindle positioning in human cells

Correct spindle positioning is fundamental for proper cell division during development and in stem cell lineages. Dynein and an evolutionarily conserved ternary complex (nuclear mitotic apparatus protein [NuMA]–LGN–Gα in human cells and LIN-5–GPR-1/2–Gα in Caenorhabditis elegans) are required for correct spindle positioning, but their relationship remains incompletely understood. By analyzing fixed specimens and conducting live-imaging experiments, we uncovered that appropriate levels of ternary complex components are critical for dynein-dependent spindle positioning in HeLa cells and C. elegans embryos. Moreover, using mutant versions of Gα in both systems, we established that dynein acts at the membrane to direct spindle positioning. Importantly, we identified a region within NuMA that mediates association with dynein. By using this region to target dynein to the plasma membrane, we demonstrated that the mere presence of dynein at that location is sufficient to direct spindle positioning in HeLa cells. Overall, we propose a model in which the ternary complex serves to anchor dynein at the plasma membrane to ensure correct spindle positioning.     

In vitro reassembly of the membranous vesicle from Escherichia coli outer membrane components: Role of individual compnents and magnesium ions in reassembly

A method was developed for the reassembly of membranous vesicle from the sodium dcoxycholate-dissociated outer membrane components of Escherichia coli. The removal of the detergent by dialysis and the presence of Mg²⁺ were essential for the reassembly.Membrane protein alone did not form any membranous structure. Closed membranous vesicles similar to the native outer membrane were reassembled only when protein was mixed with both lipopolysaccharide and phospholipid in deoxycholate solution and subsequently dialyzed. The membrane showed a distinct trilaminar structure with a center-to-center distance between two dark lines of 53 Å, which is a characteristic of the native outer membrane. This characteristic trilaminar structure was shown to be due to the presence of lipopolysaccharide. Phospholipd was required for the vesicularization of membrane. Lipopolysaccharide and/or phospholipid formed a membranous structure in the absence of protein, while the morphology of their negatively stained sample was quite different from that of the native outer membrane unless the outer membrane protein was added to the reassembly mixture.The protein from the cytoplasmic membrane was unable to reform membranous vesicle with lipopolysaccharide and phospholipid, indicating that the reassembly system discriminated outer membrane proteins from cytoplasmic membrane proteins.     

Fine structure of the tegumentary glands secreting the protective "shield" in a sessile insect (Homoptera, Diaspiddae)

The tegumentary pygidial glands of Aonidiella aurantii (Homoptera, Diaspididae) produce a secretion forming the shield of these fixed parasites of plants. They are formed of seven cells: a principal unpaired secretory cell which produces an abundant glycoproteinaceous secretion; a small associated cell with a secondary reservoir for this secretion; two accessory secretory cells which have very abundant tubular extensions coming from the plasma membrane, and a flocculent secretion gathered in a large sub-cuticular space; two cells forming an enlarged part of the excretory canal, functioning like a spinneret; and finally a single cell forming the tubular duct of this complex gland. The cuticle of the secretory cells has a very special porous structure, through which the secretion passes. The final product is a ribbon formed by two hollow strands stuck together. The exact nature of this secretion is not clear. It is comparable to a silk secretion though it has its own particular characteristics.     

Ultrastructure of Thiothrix spp. and "Type 021N" Bacteria

The ultrastructural features of two groups of filamentous sulfur bacteria, Thiothrix spp. and an unnamed organism designated "type 021N," were examined by transmission electron microscopy. Negative staining of whole cells and filaments with uranyl acetate revealed the presence of tufts of fimbriae located at the ends of individual gonidia of Thiothrix sp. strain A1 and "type 021N" strain N7. Holdfast material present at the center of mature rosettes was observed in thin sections stained with ruthenium red. A clearly defined sheath enveloped the trichomes of two of three Thiothrix strains but was absent from "type 021N" filaments. The outer cell wall appeared more complex in "type 021N" strains than in Thiothrix isolates. Bulbs or clusters of irregularly shaped cells, often present in filaments of "type 021N" bacteria, appeared to result from crosswalls which formed at angles oblique to the filament axis. The multicellular nature of these sulfur bacteria was apparent in that only the cytoplasmic membrane and peptidoglycan layer of the cell wall were involved in the septation process. Sulfur inclusions which developed in the presence of sodium thiosulfate were enclosed by a single-layered envelope and located within invaginations of the cytoplasmic membrane.     

NEW MEMBRANE FORMATION DURING CYTOKINESIS IN NORMAL AND CYTOCHALASIN B-TREATED EGGS OF XENOPUS LAEVIS : I. Electron Microscope Observations

A method is described for measuring and calculating the preexisting surface in uncleaved Xenopus eggs and the rate of surface growth in cleaving eggs. Surface-marking experiments with cytochalasin B-treated eggs show that the unpigmented surface grows by de novo formation and not by expansion of preexisting pigmented surface. The onset of new surface formation during first cleavage was studied by using transmission electron microscope and scanning electron microscope techniques. At 3–4 min and at 7–8 min after the onset of cleavage the eggs were fixed in the presence of ruthenium red (RR). Evidence is presented that unpigmented surface representing new membrane comes into appearance between four and eight min. This surface has a selective binding capacity for RR. Concomitantly with the appearance of new membrane, endoplasmic reticulum (ER) cisternae are in continuity with, and dense cytoplasmic inclusions coalesce with, the membrane along the furrow. The latter give rise to liposome-like bodies. The possibility is discussed that the ER cisternae transport a surface exudate (a carbohydrate complex), that the dense cytoplasmic inclusions represent pools of membrane precursor, and that membranogenesis takes place by direct insertion of pooled precursors into the cell surface. In a second paper, these findings will be correlated with electrophysiological measurements.     

THE FINE STRUCTURE OF CHONDROCOCCUS COLUMNARIS : III. The Surface Layers of Chondrococcus columnaris

An electron microscope study of the myxobacterium Chondrococcus columnaris has revealed the following structures in the peripheral layers of the cells: (1) a plasma membrane, (2) a single dense layer (probably the mucopeptide component of the cell wall), (3) peripheral fibrils, (4) an outer membrane, and (5) a material coating the surfaces of the cells which could be stained with the dye ruthenium red.The ruthenium red-positive material is probably an acid mucopolysaccharide and may be involved in the adhesive properties of the cells. The outer membrane and plasma membrane both have the appearance of unit membranes: an electron-translucent layer sandwiched between two electron-opaque layers. The peripheral fibrils span the gap between the outer membrane and the mucopeptide layer, a distance of about 100 A, and run parallel to each other along the length of the cell. The fibrils appear to be continuous across the ends of the cells. The location of these fibrillar structures suggests that they may play a role in the gliding motility of these bacteria.     

The Ruthenium Complex cis-(Dichloro)tetraammineruthenium(III) Chloride Presents Selective Cytotoxicity Against Murine B Cell Lymphoma (A-20), Murine Ascitic Sarcoma 180 (S-180), Human Breast Adenocarcinoma (SK-BR-3), and Human T Cell Leukemia (Jurkat) Tumor Cell Lines

The aim of present study was to verify the in vitro antitumor activity of a ruthenium complex, cis-(dichloro)tetraammineruthenium(III) chloride (cis-[RuCl₂(NH₃)₄]Cl) toward different tumor cell lines. The antitumor studies showed that ruthenium(III) complex presents a relevant cytotoxic activity against murine B cell lymphoma (A-20), murine ascitic sarcoma 180 (S-180), human breast adenocarcinoma (SK-BR-3), and human T cell leukemia (Jurkat) cell lines and a very low cytotoxicity toward human peripheral blood mononuclear cells. The ruthenium(III) complex decreased the fraction of tumor cells in G0/G1 and/or G2-M phases, indicating that this compound may act on resting/early entering G0/G1 cells and/or precycling G2-M cells. The cytotoxic activity of a high concentration (2 mg mL^?1) of cis-[RuCl₂(NH₃)₄]Cl toward Jurkat cells correlated with an increased number of annexin V-positive cells and also the presence of DNA fragmentation, suggesting that this compound induces apoptosis in tumor cells. The development of new antineoplastic medications demands adequate knowledge in order to avoid inefficient or toxic treatments. Thus, a mechanistic understanding of how metal complexes achieve their activities is crucial to their clinical success and to the rational design of new compounds with improved potency.     

Lipophilic ruthenium complexes with tuned cell membrane affinity and photoactivated uptake

Ruthenium dipyridophenazine (dppz) complexes are virtually non-emissive in aqueous solutions but show strong luminescence in hydrophobic environments, making them interesting as molecular probes in cellular imaging. We show by luminescence spectroscopy that by substituting the dppz ligand with alkyl ether chains of increasing length the complexes can be tuned from preferential intercalation into DNA to insertion in model phospholipid membranes. Confocal laser scanning microscopy (CLSM) on methanol fixed CHO-K1 cells show an analogous distribution in the cell, where the least hydrophobic complex exclusively stains the nucleus whereas the more hydrophobic ones seem to predominantly stain membrane structures in the cytoplasm. In live cells CLSM show that initially only the more hydrophobic derivatives stain the plasma membrane. However, brief further exposure to the laser light causes permeabilization of the membrane and accumulation of extracellular ruthenium complexes in internal cellular structures, similarly to the distribution found in fixed cells.     

Photocytotoxic activity of a nitrosyl phthalocyanine ruthenium complex--a system capable of producing nitric oxide and singlet oxygen

The synthesis, structural aspects, pharmacological assays, and in vitro photoinduced cytotoxic properties of [Ru(NO)(ONO)(pc)] (pc = phthalocyanine) are described. Its biological effect on the B16F10 cell line was studied in the presence and absence of visible light irradiation. At comparable irradiation levels, [Ru(NO)(ONO)(pc)] was more effective than [Ru(pc)] at inhibiting cell growth, suggesting that occurrence of nitric oxide release following singlet oxygen production upon light irradiation may be an important mechanism by which the nitrosyl ruthenium complex exhibits enhanced biological activity in cells. Following visible light activation, the [Ru(NO)(ONO)(pc)] complex displayed increased potency in B16F10 cells upon modifications to the photoinduced dose; indeed, enhanced potency was detected when the nitrosyl ruthenium complex was encapsulated in a drug delivery system. The liposome containing the [Ru(NO)(ONO)(pc)] complex was over 25% more active than the corresponding ruthenium complex in phosphate buffer solution. The activity of the complex was directly proportional to the ruthenium amount present inside the cell, as determined by inductively coupled plasma mass spectroscopy. Flow cytometry analysis revealed that the photocytotoxic activity was mainly due to apoptosis. Furthermore, the vasorelaxation induced by [Ru(NO)(ONO)(pc)], proposed as NO carrier, was studied in rat isolated aorta. The observed vasodilation was concentration-dependent. Taken together, the present findings demonstrate that the [Ru(NO)(ONO)(pc)] complex induces vascular relaxation and could be a potent anti-tumor agent. Nitric oxide release following singlet oxygen production upon visible light irradiation on a nitrosyl ruthenium complex produces two radicals and may elicit phototoxic responses that may find useful applications in photodynamic therapy.     

Structure and Composition of the Fusion Pore

Earlier studies using atomic force microscopy (AFM) demonstrated the presence of fusion pores at the cell plasma membrane in a number of live secretory cells, revealing their morphology and dynamics at nm resolution and in real time. Fusion pores were stable structures at the cell plasma membrane where secretory vesicles dock and fuse to release vesicular contents. In the present study, transmission electron microscopy confirms the presence of fusion pores and reveals their detailed structure and association with membrane-bound secretory vesicles in pancreatic acinar cells. Immunochemical studies demonstrated that t-SNAREs, NSF, actin, vimentin, α-fodrin and the calcium channels α1c and β3 are associated with the fusion complex. The localization and possible arrangement of SNAREs at the fusion pore are further demonstrated from combined AFM, immunoAFM, and electrophysiological measurements. These studies reveal the fusion pore or porosome to be a cup-shaped lipoprotein structure, the base of which has t-SNAREs and allows for docking and release of secretory products from membrane-bound vesicles.     

Properties of Membranes from the Halophyte Suaeda maritima: I. CYTOCHEMICAL STAINING OF MEMBRANES IN RELATION TO THE VALIDITY OF MEMBRANE MARKERS

The fine structure and certain cytochemical properties of theshoots from the halophyte Suaeda maritima have been examined.No major fine structural differences were observed between plantsgrown in the presence or absence of salt. Considerable evidenceof vesiculation was observed, particularly in cells of the stelartissue from the stems. The specificity of the phosphotungsticacid-chromic acid stain for plasma membranes was examined andsome doubt raised as to its widespread applicability as a plasmamembrane marker in plant cells. The suitability of other membranemarkers such as ruthenium red and ATPase activity were investigated.     

THE FINE STRUCTURE OF CHONDROCOCCUS COLUMNARIS : I. Structure and Formation of Mesosomes

Cells of Chondrococcus columnaris were sectioned and examined in the electron microscope after fixation by two different methods. After fixation with osmium tetroxide alone, the surface layers of the cells consisted of a plasma membrane, a dense layer (mucopeptide layer), and an outer unit membrane. The outer membrane appeared distorted and was widely separated from the rest of the cell. The intracytoplasmic membranes (mesosomes) appeared as convoluted tubules packaged up within the cytoplasm by a unit membrane. The unit membrane surrounding the tubules was continuous with the plasma membrane. When the cells were fixed with glutaraldehyde prior to fixation with osmium tetroxide, the outer membrane was not distorted and separated from the rest of the cell, structural elements (peripheral fibrils) were seen situated between the outer membrane and dense layer, and the mesosomes appeared as highly organized structures produced by the invagination and proliferation of the plasma membrane. The mesosomes were made up of a series of compound membranes bounded by unit membranes. The compound membranes were formed by the union of two unit membranes along their cytoplasmic surfaces.