Subunit arrangement of Escherichia coli F1-ATPase studied by scanning transmission electron microscopy

The shape and the arrangement of subunits in Escherichia coli F1-ATPase (ECF1) lacking the delta subunit have been explored with a high performance scanning transmission electron microscope. In tilting experiments, the ECF1 molecule appeared as a flat cylinder whose width (approx. 120 A) was about twice its height. The symmetry of front view projections of ECF1 has been investigated by computer analysis. In a population taken at random from the data bank, one third of the particles showed five-fold radial symmetry components, one third six-fold radial symmetry components and the last third no typical symmetry. The six-fold radial symmetry was consistent with a hexagonal arrangement of six large peripheric masses, which probably correspond to the three alpha and the three beta subunits of ECF1. The five-fold radial symmetry was tentatively explained by a fusion of two juxtaposed peripheric subunits. Lateral projections showed a zig-zag organization of the large masses, suggesting that the large alpha and beta subunits are located on two levels, with some degree of intercalation between the subunits of the two levels.     

Conformational analysis of 16 S ribosomal RNA from Escherichia coli by scanning transmission electron microscopy

Digitized images of molecules of 16 S rRNA from Escherichia coli, obtained by scanning transmission electron microscopy (STEM), provide quantitative structural information that is lacking in conventional electron micrographs. We have determined the morphology, total molecular mass, mass distribution within individual rRNA molecules and apparent radii of gyration. From the linear density (M/L) we have assessed the number of strands in the structural backbone of rRNA and studied the pattern of branching and folding related to the secondary and tertiary structure of rRNAs under various buffer conditions. Even in reconstitution buffer 16 S RNA did not show any resemblance to the native 30 S subunit.     

Direct visualization of intracellular calcium in rat osteoblasts by energy-filtering transmission electron microscopy

Osteoblasts are the highly specialized bone cells responsible for matrix mineralization. Mineralization is a complex, incompletely understood, process involving intracellular calcium homeostasis. Rapid changes in ionized calcium concentration ([Ca²⁺]_i) occur in these cells, but the intracellular distribution of total calcium, which may be involved in matrix mineralization, remains unknown. We have therefore investigated the distribution of total calcium in osteoblasts either ex vivo from rapidly mineralizing neonatal rat bones or in the same cells cultured to confluence before they had entered the mineralization phase, and without stimulation for mineralized matrix formation. All cells were examined bone-untreated (controls) or following the addition of the ionophore ionomycin that induced a large and sustained increase in [Ca²⁺]_i. Cryomethods, quick-freezing and freeze-drying, and OsO₄ vapor fixation were employed to preserve the original calcium distribution, and the preservation was verified by secondary ion mass spectrometry (SIMS). Intracellular calcium distribution was identified by energy-filtering transmission electron microscopy (EELS). Scarce calcium signals were recorded from all osteoblasts maintained in buffer (controls). Ionomycin addition resulted in the accumulation of calcium in mitochondria, and more calcium was stored in the mitochondria of osteoblasts involved in mineralization than in those of osteoblasts before mineralization. Moreover, in the former, strong calcium signals were recorded around the junctions between mitochondria and the endoplasmic reticulum. Thus EELS allowed to obtain high-resolution total calcium maps in defined intracellular structures, but only at elevated calcium levels.     

Heterogeneity of Escherichia coli ribosomes established by scanning transmission electron microscopy.

Quantitative mass image analysis of Escherichia coli ribosomal particles by scanning transmission electron microscopy (STEM) provided direct evidence that presumably homogeneous preparations of ribosomes are, in reality, populations of heterogeneous particles. Variations in composition, relative molecular mass (Mr) and shape were observed both in the monosomes and in the ribosomal subunits. None of these changes can be resolved visually; they can be evaluated only by computer processing. The variations in relative mass and shape monitored by values of radius of gyration (RG) were attributed to the loss of ribosomal proteins and/or factors and correlated with the changes in ribosome composition and biological activity. The highest activity was found in monosomes prepared from the standard 0.5 M NH4Cl wash. With increasing concentrations (up to 1.5 M) of NH4Cl in the wash buffer the activity decreased slowly, then dropped rapidly to about half in 2 M NH4Cl. The most striking effects were observed in ribosomal particles washed with 0.1 M NH4Cl. The 70S monosomes and the 30S subunits attained maximum Mr and RG values (2660 kDa and 76 A, and 990 kDa and 75 A, respectively), which were greater than the theoretical values, while the activity was minimal (approximately 12%). The Mr and RG parameters of the 50S subunits remained uneffected by the NH4Cl washes (approximately 1600 kDa and 68 A).     

Cytochemical techniques and energy-filtering transmission electron microscopy applied to the study of parasitic protozoa

The study of parasitic protozoa plays a major role in cell biology, biochemistry and molecular biology. Numerous cytochemical techniques have been developed in order to unequivocally identify the nature of subcellular compartments. Enzyme and immuno-cytochemistry allow the detection of, respectively, enzymatic activity products and antigens in particular sites within the cell. Energy-filtering transmission electron microscopy permits the detection of specific elements within such compartments. These approaches are particularly useful for studies employing antimicrobial agents where cellular compartments may be destroyed or remarkably altered and thus hardly identified by standard methods of observation. In this regard cytochemical and spectroscopic techniques provide valuable data allowing the determination of the mechanisms of action of such compounds. Published: August 4, 2001     

The structure of Escherichia coli signal recognition particle revealed by scanning transmission electron microscopy

Structural studies on various domains of the ribonucleoprotein signal recognition particle (SRP) have not converged on a single complete structure of bacterial SRP consistent with the biochemistry of the particle. We obtained a three-dimensional structure for Escherichia coli SRP by cryoscanning transmission electron microscopy and mapped the internal RNA by electron spectroscopic imaging. Crystallographic data were fit into the SRP reconstruction, and although the resulting model differed from previous models, they could be rationalized by movement through an interdomain linker of Ffh, the protein component of SRP. Fluorescence resonance energy transfer experiments determined interdomain distances that were consistent with our model of SRP. Docking our model onto the bacterial ribosome suggests a mechanism for signal recognition involving interdomain movement of Ffh into and out of the nascent chain exit site and suggests how SRP could interact and/or compete with the ribosome-bound chaperone, trigger factor, for a nascent chain during translation.     

The quaternary structure of Escherichia coli RNA polymerase studied with (scanning) transmission (immuno)electron microscopy

A model for the quaternary structure of Escherichia coli RNA polymerase (nucleosidetriphosphate:RNA nucleotidyltransferase, EC 2.7.7.6) is presented. It is based on results from classification of profiles of enzyme molecules, and from application of immuno electron microscopy. Classification of molecules, prepared with the single carbon layer technique, was first achieved for images recorded in dark field with the scanning transmission electron microscope and later on for images recorded in bright-field transmission electron microscopy. It results in five approximately equally sized groups, containing about 80% of the core enzyme profiles. Holoenzyme profiles can be grouped into the same classes, and have approximately the same dimensions (9 nm X 16 nm). Based on the shapes and sizes of the classified profiles, a tentative model for core enzyme has been constructed. Correlation of shadow projections of this model, with the distributions of attachment sites of antibodies against alpha, beta, beta' and sigma over the profiles, has led to models for core and holoenzyme in which the subunits are localized. The model is compared with literature data on the quaternary structure of RNA polymerase.     

Use of energy-filtering transmission electron microscopy for routine ultrastructural analysis of high-pressure-frozen or chemically fixed plant cells

Summary. In the present study energy-filtering transmission electron microscopy by use of an in-column spectrometer is employed as a powerful tool for ultrastructural analysis of plant cells. Images of unstained very thin (50 nm) and thick (140 nm) sections of the unicellular green alga Micrasterias denticulata, as a model system for a growing plant cell, taken by conventional transmission electron microscopy are compared to those obtained from filtering at zero energy loss (elastic bright field) and to those generated by energy filtering below the carbon-specific absorption edge at about 250 eV. The results show that the high-contrast images produced by the latter technique are distinctly superior in contrast and information content to micrographs taken at conventional transmission electron microscopy mode or at elastic bright field. Post- or en bloc staining with heavy metals, which is indispensable for conventional bright-field transmission electron microscopy, can be completely omitted. Delicate structural details such as membranous or filamentous connections between organelles, organelle interactions, or vesicle and vacuole contents are clearly outlined against the cytoplasmic background. Also, immunoelectron microscopic localization of macromolecules benefits from energy-filtering transmission electron microscopy by a better and more accurate assignment of antigens and structures and by facilitating the detection of immunomarkers without renunciation of contrast.     

Membrane turnover in crab photoreceptors studied by high-resolution scanning electron microscopy and by a new technique of thick-section transmission electron microscopy

Summary Crab photoreceptors were examined after treatment by the osmium-DMSO-osmium method for high-resolution scanning electron microscopy. This technique of specimen preparation was also adapted for transmission electron microscopy, enabling sections up to 1 urn thick to be viewed in a conventional microscope at 75 kV. With appropriate pretreatment, some cytoskeletal elements can be visualised by both techniques. The methods were then used to investigate some of the daily changes known to occur in photoreceptor cell structure. Striking differences were found in the structure of Golgi bodies present in retinula cells during the synthesis and breakdown phases of the daily cycle of photoreceptor membrane turnover. Cyclic changes were also noticed in the mitochondria of retinula cells, and additional evidence was found for a previously proposed model of rhabdomeral microvillus formation.     

The monomeric arrangement in the dimer of Escherichia coli RNA polymerase holoenzyme studied with (scanning) transmission electron microscopy

A model is presented for the arrangement of the monomers in the dimer of Escherichia coli RNA polymerase holoenzyme. The dimers, formed at low salt concentration in the presence of magnesium ions, were negatively stained and photographed in TEM and STEM. The profiles of the monomers forming the dimers were classified. In 61% (134) of the dimers, both monomeric profiles could be classified. We determined the regions on the monomer which interact in the dimer, and the frequencies with which the various possible contacts between regions occur. We conclude that the binding site on the monomer is constituted by one of the α subunits and the β subunit, and that the association between the monomers is isologous.In the absence of Mg²⁺, we found that the holoenzyme exists mainly as a monomer at low salt concentration. The differences in the degree of oligomerization of RNA polymerase at low salt concentrations are discussed in view of our model.     

Visualization of ion-dependent conformational changes in Escherichia coli 23 S rRNA by scanning transmission electron microscopy

Electron micrographs of Escherichia coli 23 S rRNA molecules obtained by scanning transmission electron microscopy, unstained and under nondenaturing conditions, reveal previously unresolved structural patterns. The complexity of the pattern is dependent upon the ambient ionic strength conditions. In water and in very low ionic strength buffer, the conformation of 23 S rRNA is characterized by an extended framework, with short side branches related to the secondary and tertiary structure of the molecule. The total length of this filamentous complex is approximately 2500 A, only about one-fourth of the length of 23 S rRNA when fully stretched under the denaturing conditions used for imaging by conventional electron microscopy. These data, supplemented by the determination of the linear density (M/L), suggest that in low ionic strength the backbone of 23 S rRNA is formed by a structure corresponding, on the average, to the mass of four nucleotide strands (M/L approximately equal to 480 Da/A). With increasing ionic strength, 23 S rRNA coils into more compact forms. Molecules in these states can be characterized by apparent radii of gyration (RG), which can be calculated from the mass distribution within the digitized images of individual RNA molecules. The 23 S rRNA is in its most condensed form (RG = 115 A) in ribosomal reconstitution buffer; however, it still does not attain the compactness of the large subunit (RG = 69 A), nor does it show any resemblance to the native 50 S subunit. The net content of ordered secondary structure, as determined by circular dichroism spectroscopy, is not visibly affected by the changes of ionic strength conditions. These results imply that the observed conformational changes in 23 S rRNA are caused by intramolecular folding of the 23 S rRNA strands induced by the shielding effect of ambient charges.     

Pitfalls of immunogold labeling: analysis by light microscopy, transmission electron microscopy, and photoelectron microscopy

The immunogold method is widely used to localize, identify, and distinguish cellular antigens. There are, however, some pitfalls that can lead to nonspecific binding, particularly in cytoskeletal studies with gold probes prepared from small gold particles. We present a list of suggestions for minimizing nonspecific binding, with particular attention to two problems identified in this study. First, we find that the method used to prepare the colloidal gold particles affects the degree of nonspecific binding. Second, the standard BSA-stabilized small gold probes evidently possess exposed regions that bind to the proteins of cytoskeletal preparations. This was investigated in whole-mount cytoskeletal preparations of cultured cells by use of light microscopy, transmission electron microscopy, and photoelectron microscopy of silver-enhanced specimens. Gold probes were made from approximately 5-nm particles generated by reduction of HAuCl4 with three different reducing agents: white phosphorus, sodium borohydride, and citrate-tannic acid. All three preparations stabilized in the conventional way showed significant levels of nonspecific binding, which was highest with citrate-tannic acid. This problem was largely solved with all three types of probes by including fish gelatin in the probe buffer, by substituting fish gelatin for the BSA stabilizer used to prepare the probes, or by pre-adsorption methods. Application of these techniques resulted in clear immunogold labeling patterns with minimal nonspecific background.     

The effect of salt and pH on block liposomes studied by cryogenic transmission electron microscopy

Recently, we have reported the discovery of block liposomes (BLs), a new class of liquid (chain-melted) vesicles, formed in mixtures of the curvature-stabilizing hexadecavalent cationic lipid MVLBG2, the neutral lipid 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC), and water with no added salt. BLs consist of connected spheres, pears, tubes, or rods. Unlike in typical liposome systems, where spherical vesicles, tubular vesicles, and cylindrical micelles are separated on the macroscopic scale, shapes remain connected and are separated only on the nanometer scale within a single BL. Here, we report structural studies of the effect of salt and pH on the BL phase, carried out using differential interference contrast microscopy (DIC) and cryogenic transmission electron microscopy (cryo-TEM). Addition of salt screens the electrostatic interactions; in low-salt conditions, partial screening of electrostatic interactions leads to a shape transition from BLs to bilamellar vesicles, while in the high-salt regime, a shape transition from BLs to liposomes with spherical morphologies occurs. This demonstrates that strong electrostatic interactions are essential for BL formation. Understanding the control of liposome shape evolution is of high interest because such shape changes play an important role in many intracellular processes such as endocytosis, endoplasmatic reticulum-associated vesiculation, vesicle recycling and signaling.     

Zygote formation inCosmarium botrytis studied by scanning and transmission electron microscopy,phase-contrast,and Calcofluor staining

Summary Rapid zygote formation byCosmarium botrytis was induced in a liquid medium by incubation in 5% CO₂. Conjugation and zygote formation were studied by SEM, TEM, phase-contrast, and Calcofluor fluorescence microscopy. It was observed that the cells divided immediately prior to conjugation and formed Calcofluor fluorescent conjugation papillae as soon as the primary wall was shed. The conjugating cells and the resultant zygote were envelopped by a non-fluorescent mucilagenous envelope which was eventually pierced by the zygote spines, but never shed. The very young smooth-walled zygote had a thick Calcofluor fluorescent wall. At that stage the zygote could be plasmolysed in 0.4 M mannitol, but no protoplast could be induced to emerge even with the addition of up to 5% Cellulysin; probably indicating that the zygote wall composition and structure is different from that of the secondary wall of the vegetative cells, particularly in the absence of mucilage pores.     

Localization of ampicillin-sensitive sites in Escherichia coli by electron microscopy.

Growth of Escherichia coli B/r ATCC 12407 (doubling time, 65 to 70 min) in the presence of 500 mug of ampicillin per ml for 15 to 20 min induces a sphere alongside the cell. The position was determined with respect to the length axis of the cell by electron microscopy. Although spheres may be found anywhere, some prominent sites do occur. In the shortest cells, which have a length of about 1.5 mum, they are found at the presumed new cell pole. In slightly older cells (length, about 1.8 mum), the position of the sphere is not well defined. Later on spheres occur predominantly at the cell center. In dividing cells (average length, 2.5 mum) a sphere may also occur at about one-quarter of the cell length. The position of the spheres bears resemblance to sites where a pulse of 3H-labeled diaminopimelic acid is incorporated into the peptidoglycan, as has been found by others.     

Gap junctions in the epidermis of fetal rats studied by transmission electron microscopy

In the ventral epidermis of fetal rats the size and distribution of intercellular gap junctions changed during differentiation. In the young fetus, between 13 and 17 days, large gap junctions sometimes exceeding 3 micron in profile length were found predominantly in basal cells. As the epidermis increased in thickness the mean profile length diminished until only small gap junctions were present mainly in more superficial layers even persisting into the stratum corneum. Endocytosis of the intercellular gap junctions gave rise to intracytoplasmic annular gap junctions (AGJs) which occurred after 17 days predominantly in the superficial three layers of the epidermis. The AGJs diminished in mean diameter with the age of the fetuses possibly as a consequence of the decreasing size of the intercellular gap junctions from which they had formed. Rarely sequestration of AGJs by cytoplasmic membranes occurred but many recognizable AGJs persisted into the stratum corneum. As in other developing systems, the function of gap junctions in epidermis is unknown but the extensive junctions of younger epidermis might be related to the maintenance of a greater level of uniformity both of mitotic activity and of differentiation.     

Cellular localisation by immunolabelling and transmission electron microscopy of oxaloacetate decarboxylase or its individual subunits synthesised in Escherichia coli

Abstract The genes oadGAB encoding the oxaloacetate decarboxylase γ, α and β-subunits from Klebsiella pneumoniae were expressed in Escherichia coli . Using different expression vectors, the entire enzyme or its individual subunits were synthesised. The expression was evidenced immunologically in whole cells with polyclonal antibodies raised against the purified oxaloacetate decarboxylase. The expressed α-subunit or a combination of a and β-subunits were shown to reside in the cytoplasm, while the entire oxaloacetate decarboxylase or a γα-complex were located mostly in the cytoplasmic membrane. Interestingly, overexpression of the γα-complex or the entire oxaloacetate decarboxylase in E. coli led to a significant immunogold labelling in the cytoplasm, indicating that the a-subunit was not completely complexed to the membrane-bound γ or βγ-subunits.