Electron microscopic analysis of lymphocyte fibrillar elements during the redistribution of surface receptors

Filaments 5 nm thick, located throughout the cytoplasm mainly along the surface, are observed in intact lymphocytes. In the control glycerinized lymphocytes, besides the above filaments aggregations of filaments nearly 3 nm in diameter were found. After the treatment of cells by antimurine serum or ferritin-conjugated concanavalin A, some fibrillar structures are observed mainly in the cap region in the form of filaments 5-6 nm of thickness, radially directed towards the plasma membrane. After glycerinization, three types of filaments are observed being, respectively, near 3, 5-6 and almost 8 nm in diameter. Two latter types of filaments are decorated by S1-myosine fragments which indicates their actine nature. Differences in the character and distribution of myofibrils in the cytoplasm of intact cells and cells with caps may witness in favour of their involvement in the processes associated with redistribution of surface receptors.     

Electron microscopic analysis of membrane assemblies formed by the bacterial chemotaxis receptor Tsr

The serine receptor (Tsr) from Escherichia coli is representative of a large family of transmembrane receptor proteins that mediate bacterial chemotaxis by influencing cell motility through signal transduction pathways. Tsr and other chemotaxis receptors form patches in the inner membrane that are often localized at the poles of the bacteria. In an effort to understand the structural constraints that dictate the packing of receptors in the plane of the membrane, we have used electron microscopy to examine ordered assemblies of Tsr in membrane extracts isolated from cells engineered to overproduce the receptor. Three types of assemblies were observed: ring-like "micelles" with a radial arrangement of receptor subunits, two-dimensional crystalline arrays with approximate hexagonal symmetry, and "zippers," which are receptor bilayers that result from the antiparallel interdigitation of cytoplasmic domains. The registration among Tsr molecules in the micelle and zipper assemblies was sufficient for identification of the receptor domains and for determination of their contributions to the total receptor length. The overall result of this analysis is compatible with an atomic model of the receptor dimer that was constructed primarily from the X-ray crystal structures of the periplasmic and cytoplasmic domains. Significantly, the micelle and zipper structures were also observed in fixed, cryosectioned cells expressing the Tsr receptor at high abundance, suggesting that the modes of Tsr assembly found in vitro are relevant to the situation in the cell.     

Cholesterol modulation of nicotinic acetylcholine receptor surface mobility

Nicotinic acetylcholine receptor (AChR) function and distribution are quite sensitive to cholesterol (Chol) levels in the plasma membrane (reviewed by Barrantes in J Neurochem 103 (suppl 1):72–80, 2007). Here we combined confocal fluorescence recovery after photobleaching (FRAP) and confocal fluorescence correlation spectroscopy (FCS) to examine the mobility of the AChR and its dependence on Chol content at the cell surface of a mammalian cell line. Plasma membrane AChR exhibited limited mobility and only ~55% of the fluorescence was recovered within 10 min after photobleaching. Depletion of membrane Chol by methyl-β-cyclodextrin strongly affected the mobility of the AChR at the plasma membrane; the fraction of mobile AChR fell from 55 to 20% in Chol-depleted cells, whereas Chol enrichment by methyl-β-cyclodextrin-Chol treatment did not reduce receptor mobility at the cell surface. Actin depolymerization caused by latrunculin A partially restored receptor mobility in Chol-depleted cells. In agreement with the FRAP data, scanning FCS experiments showed that the diffusion coefficient of the AChR was about 30% lower upon Chol depletion. Taken together, these results suggest that membrane Chol modulates AChR mobility at the plasma membrane through a Chol-dependent mechanism sensitive to cortical actin.     

Electron microscopic analysis of tropomyosin paracrystals.

Negatively stained images of divalent cation-induced tropomyosin paracrystals show polymorphic patterns which are almost bipolar. Although these bipolar forms are naturally due to antiparallel arrays of molecules, the precise molecular arrangements have not been clarified yet except in the case of one type of these polymorphic paracrystals by Stewart and McLachlan [(1976) J. Mol. Biol. 103, 251--269]. In the previous paper we showed that the lead-induced polar paracrystal is a parallel and in-register array of tropomyosin molecules. Moreover, we have made it possible to locate a given residue on the staining pattern. By overlapping two photographic transparencies of the polar paracrystal antiparallel, directly observed images of polymorphic bipolar paracrystals could be synthesized photographically with fidelity. The overlap length between N-terminals of antiparallel pairs of molecules could be easily determined without any assumptions. Next, we considered the stabilizing forces involved in the morphogenesis of such polymorphic paracrystals. The cation-bridged attractive forces already proposed by some groups were insufficient to account for the stability of some specific forms of tropomyosin paracrystals. From the primary amino acid sequence of tropomyosin, we calculated the changes of repulsive forces between the basic residues with changes of molecular overlap length between the N-terminals of antiparallel pairs. By setting the values of charge appropriately, we could account well for the stability of the polymorphic structures observed by electron microscopy.     

Electron microscopic study of chromatin in maturing lymphocyte nuclei using ammoniacal silver]

By means of the ammoniacal silver reaction, cytochemical properties of the chromatin in white rat lymph node lymphocytes were investigated at different stages of their maturation. Electron dense granules of the reaction product are shown to be localized over the compact chromatin region. The number of granules increases as the amount of compact chromatin enlarges. A possible role of arginine histones in the process of chromatin condensation is suggested, this suggestion being based on the assumption that ammoniacal silver binds with active arginine groups of histones.     

Electron microscopic observation of mu-opioid receptor in the rat area postrema.

A simple preembedding avidin-biotin-peroxidase complex technique was used to study the ultrastructural localization of mu-opioid receptor in the rat area postrema. By using low concentrations of the first antiserum for incubation with a short reaction time to 3,3'-diaminobenzidine, the immunostaining was faint at the light microscopic level. However, at the electron microscopic level, strong immunoreaction was observed. Mu-Opioid receptors were found to be localized on the postsynaptic membrane of dendrites, extrasynaptic plasma membrane, and the surface of the small, clear vesicles in axon terminals. Of the total 283 immunopositive profiles observed, 68.2% (193 of 283) were dendrites, 29.3% (83 of 283) were axon terminals, and 2.5% (7 of 283) were myelinated axons. No immunostained neuron bodies were found in the present study; 109 mu-opioid receptor immunoreactive dendrites received synapses (56.5%, 109 of 193) from nonimmunoreactive (84.4%, 92 of 109) or immunoreactive (15.6%, 17 of 109) axon terminals, whereas 84 dendrites (43.5%, 84 of 193) were found without receiving synapses. The present study shows that the mu-opioid receptor in the area postrema plays a role mainly at the synapses.     

Electron microscopic study of the interrelations of the bush-like receptor terminals with the tissue

A study was made of the ultrastructure of elements linking the bush-like receptors with tissues of the frog bladder. It is demonstrated that the connection is realized mainly by collagen fibres, whose dense and irregular net is branching the nervous terminals. Besides, a dense contact has been found between the terminals and epithelial cells without any layer of collagen fibres. No desmosomal connection between terminals and tissue elements was found. It is suggested that the above peculiarities of the connections may influence considerably the functional-adaptational characteristics of the receptors.     

Electron microscopic and preparative methods for the analysis of isopod cuticle

The crustacean cuticle consists of a complex organic matrix and a mineral phase. The physical and chemical properties of the cuticle are corellated to the specific functions of cuticular elements, leading to a large variety in its structure and composition. Investigation of the structure-function relationship in crustacean cuticle requires sophisticated methodological tools for the analysis of different aspects of the cuticular architecture. In the present paper we report improved preparation methods that, in combination with various electron microscopic techniques, have led to new insights of cuticle structure and composition in the tergite cuticle of Porcellio scaber. We used thin sections of non-decalcified tergites and decalcified resin embedded material for transmission electron microscopy and scanning transmission electron microscopy. Etched sagittal planes of bulk tergite samples were analysed with field emission scanning electron microscopy. We have found a distinct distal region within the exocuticle that differs from the subjacent proximal exocuticle in the arrangement of fibres. Within this distal exocuticle chitin-protein fibrils assemble to fibres with diameters between 15 and 50 nm that are embedded in a mineral matrix. In the proximal exocuticle and the endocuticle fibrils do not assemble to fibres and are surrounded by mineral individually. Furthermore, we show that the pore canals are filled with mineral, and demonstrate that mild etching of polished sagittal cuticle surfaces reveals regions containing mineral of diverse solubility.     

Electron microscopic morphometry

The fundamental concepts of morphometry, the principal correction factors for systematic errors and the basic principles of efficient sampling are outlined for quantitative morphology at the electron microscopic level of resolution. The important usefulness of correlating electron microscopic morphometry with complementary light microscopic morphometry is emphasized.     

Electron microscopic analysis of the structure of metaphase chromosomes after liposome treatment

The fine structure of the methaphase chromosomes from the Chinese hamster cell culture was studied after the incubation with lyposomes isolated from hen egg yolk phosphatidilcholine, or from the total rat liver phospholipid. It has been found that during incubation lyposomes surround chromosomes, form a multifold covering considerably decondensing the chromosome matrix. The data obtained indicate that the analogues of cell membrane - lyposomes - may be involved in the regulation of the structural organization of metaphase chromosomes.     

Electron microscopic analysis of replicating DNA of sea urchin embryos

DNA was extracted from Paracentrotus lividus embryos at the third S phase after fertilization and analyzed with the electron microscope. The most relevant structures observed in this actively replicating DNA are clusters of short, closely spaced microbubbles (about 0.1 μm long on the average), partially or entirely single-stranded molecules and few linear forks. Unexpectedly, no long eye forms were observed. The analysis of DNA purified from gastrulae and from adult somatic tissues has revealed the same structures, although at a low frequency. A quantitative analysis has been carried out to determine the size distribution and spacing of microbubbles. A number of control experiments have been performed to characterize these structures better. Various possibilities are discussed to account for the presence of the observed forms and the absence of larger eyes.     

Electron microscopic analysis of the yeast mitochondrial DNA segment conferring chloramphenicol resistance

Summary Mitochondrial DNAs from six ^- mutants carrying the genetic locus Ribl and deleted for the rest of the genome were analyzed. Distribution of circular molecules from one mutant followed exactly the frequency rule, 1/n, for multimers with discreet classes n, 2n, 3n, etc. Another, genetically unstable mutant displayed a continuous spectrum of circular molecules of various lengths. Four other mutants contained multiple series of circular molecules. Partial denaturation maps show that the mutants analyzed show a common segment ca. 1.0 m long and differ by characteristic deletions of extremites of this segment. Short terminal deletions of the right i.e. pointing towards the Rib3 locus, terminus of this segment are correlated with modifications of the recombination properties related to the locus.