The molecular structure of human erythrocyte spectrin. Biophysical and electron microscopic studies.

Molecules of human erythrocyte spectrin have been examined by electron microscopy after low-angle shadowing. Spectrin heterodimers and tetramers were first purified and characterized by polyacrylamide gel electrophoresis and analytical ultracentrifugation under conditions which minimize proteolysis and aggregation. The heterodimers and tetramere were separated for low-angle shadowing by gel filtration in ammonium acetate buffer at physiological ionic strength, in which they showed sedimentation coefficients of 8.9 S and 12.5 S, respectively, similar to those values reported for heterodimers and tetramers in non-volatile buffers. The ammonium acetate buffer promoted the dissociation of spectrin tetramers into heterodimers under conditions in which tetramers in NaCl or KCl buffers are stable. When visualized by low-angle unidirectional and rotary shadowing, spectrin heterodimers appeared as long flexible molecules with a mean shadowed length of 97 nm. Each heterodimer, composed of the two polypeptide chains, band 1 (240,000 M_r) and band 2 (220,000 M_r), often appeared as two separate strands which lay partially separated from one another or coiled round each other in a loose double helix. The association between these polypeptides appears to be weak, except at both ends of the molecule where there are sites of strong binding. Tetramers are formed by the end-to-end association of two spectrin heterodimer molecules without measurable overlap, and have a mean shadowed length of 194 nm. This association to form tetramers probably involves head-to-head binding of the heterodimers, since the higher oligomers to be expected from a head-to-tail binding mode are not observed. The molecular shape of spectrin is quite distinct from that of myosin, to which it has often been likened.     

Correlative scanning-transmission electron microscopic examination of the perinatal rat brain

Summary The ultrastructural organization of the perinatal hypothalamus and the dynamics of neuronal and ependymal growth and plasticity were examined in this investigation. The brains of fetal rats 16, 17 and 18 days in utero and those of postnatal rats 1–16 days post partum were fixed with aldehyde fixatives and prepared for combined SEM/TEM analysis. By day 17 in utero the ventricular (ependymal) surfaces of the fetal thalamic wall, cerebral vesicle and rhomboid fossa were relatively well differentiated with cilia and microvilli. Type II histiocytes were the first supraependymal cell to appear upon the ventricular lumen and were evident by day 17 in utero. In contrast, the apical surfaces of tanycytes of the infundibular recess as well as those of most other circumventricular organs were poorly differentiated and unremarkable. Tanycytes of the infundibular recess exhibited a simple hexagonal mosaic pattern of apposed plasmalemmata and even by day 1 post partum few cilia or microvilli were evident.By day 5–6 post partum Type I supraependymal neurons and axonal processes began to make their appearance with some emerging from the underlying parenchyma of the median eminence. By day 16 post partum the ventricular surface of the infundibular recess was comparable with that of the adult.The Type I supraependymal neurons are remarkably similar in their ultrastructural organization with parvicellular neurosecretory neurons elsewhere in the endocrine hypothalamus. Their emergence at day 5–6 post partum suggests a possible correlation with the critical period of sexual differentiation and a potential receptor role for this cell line. On the contrary this phenomenon may simply be a developmental anomaly. Nonetheless, the mergence of such elements upon the lumen of the third cerebral ventricle underscores a remarkable degree of neuronal plasticity in the perinatal hypothalamus.Supported by USPHS Program Project Grant NS 11642-04 and USPHS-BRSG Grant RR-05403.The authors wish to thank N. Kutryeff for her excellent technical assistance     

Isolation and electron microscopic observations of intracytoplasmic inclusions containing Chlamydia psittaci.

Intracytoplasmic inclusions containing Chlamydia psittaci were isolated by a newly established method. Infected L-cells at 20 h after infection were suspended in 0.25 M sucrose-tris(hydroxymethyl)aminomethane buffer containing ethylene-diaminetetraacetic acid, homogenized in a Dounce tissue grinder, and filtered through a 2,000-mesh screen. Isolated inclusions were stabilized in 5% bovine serum albumin in 10 mM tris(hydroxymethyl)aminomethane buffer. Electron microscopic observations revealed the presence of surface projections on the vegetative, reticulate bodies and a direct connection between the reticulate bodies and the inclusion membrane by means of projections.     

Scanning electron microscopic examination of the secondary constriction of Vero cells

Vero cells (African green monkey kidney in origin) were prepared by the conventional air-drying method and then processed for SEM by a modification of the conductive method based on thiocarbohydrazide-osmium binding [3]. Under SEM, not only metaphase chromosomes but also resting nuclei showed distinct fibres 30 nm in diameter. A few such fibres were found to run across the secondary constriction of the NOR-carrying chromosome.     

Alterations to the cytoskeleton of erythrocytes infected with frog erythrocytic virus: a fluorescence and electron microscopic study.

Erythrocytes of bullfrogs (Rana catesbeiana) infected with frog erythrocytic virus are spheroid and their nucleus is displaced. In contrast, uninfected cells are ellipsoid and have a centralized nucleus. Fluorescent staining revealed that these changes are correlated with alterations to components of the erythrocyte cytoskeleton. Uninfected erythrocytes contained a broad, continuous marginal band of microtubules, which appeared thinner and interrupted in infected cells. The described disruption of microtubules was associated with an inability to polymerize the tubulin pool with the addition of 12 microM taxol. The arrangement of submembranous microfilaments in uninfected erythrocytes was not significantly altered in infected cells. Vimentin filaments were distributed throughout the cytoplasm and around the nucleus of uninfected cells, and concentrated at the cell and nuclear peripheries. Cytoplasmic pockets that did not contain vimentin filaments were associated with the viral assembly site(s) in infected cells. These data suggest that the distortion of viral-infected erythrocytes could be due, in part, to an irreversible depolymerization of microtubules of the marginal band and a reorganization of the vimentin filament network.     

Calmodulin-binding protein of erythrocyte cytoskeleton

Previously we have shown that purified spectrin binds calmodulin in the presence of Ca²⁺ with a Kd value of 3 μM (Sobue, K. et al. (1980) Biochemistry International 1, 561–566). We now provide evidence that the calmodulin-binding activity found in the human erythrocyte cytoskeleton is indeed due to spectrin and no other binding proteins are involved, i.e. the binding activity was purified from the erythrocyte cytoskeleton quantitatively and the purified peak contained spectrin as the only protein constituent. Moreover, Kd value (2.8 μM) and the maximum binding capacity (160,000 – 200,000 calmodulin per cell) obtained from the kinetic analysis of the binding activity in the crude cytoskeleton agreed with the corresponding values reported for purified spectrin. Since the concentration of calmodulin in the erythrocyte cell, which was 2.5 μM or 1.6 × 10⁵ molecules per cell, is close to both the Kd value and the number of the binding sites in the cell, respectively, free calmodulin in the erythrocyte cell may be in a dynamic equilibrium with the spectrin-bound form in vivo depending upon the intracellular concentration of Ca²⁺.     

Computer simulation of a model network for the erythrocyte cytoskeleton.

The geometry and mechanical properties of the human erythrocyte membrane cytoskeleton are investigated by a computer simulation in which the cytoskeleton is represented by a network of polymer chains. Four elastic moduli as well as the area and thickness are predicted for the chain network as a function of temperature and the number of segments in each chain. Comparisons are made with mean field arguments to examine the importance of steric interactions in determining network properties. Applied to the red blood cell, the simulation predicts that in the bilayer plane the membrane cytoskeleton has a shear modulus of 10 +/- 2 x 10(-6) J/m2 and an areal compression modulus of 17 +/- 2 x 10(-6) J/m2. The volume compression modulus and the transverse Young's modulus of the cytoskeleton are predicted to be 1.2 +/- 0.1 x 10(3) J/m3 and 2.0 +/- 0.1 x 10(3) J/m3, respectively. Elements of the cytoskeleton are predicted to have a mean displacement from the bilayer plane of 15 nm. The simulation agrees with some, but not all, of the shear modulus measurements. The other predicted moduli have not been measured.     

Oligomeric states of spectrin in normal erythrocyte membranes: Biochemical and electron microscopic studies

We estimated the relative amounts of oligomeric species of spectrin in 0°C red-cell-membrane extracts, including those released from spectrin-actinpolypeptide 4.1 complexes after mild urea treatment. Spectrin dimers, tetramers, and medium-size oligomers were the prominent species, accounting for 5%–10%, 45%–55%, and 25%–35% of spectrin, respectively. When examined by low-angle rotaryshadowing electron microscopy, these medium-size spectrin oligomers (e.g., hexamers, octamers, decamers, dodecamers, and quadecamers) appeared as polyskelions formed by head-to-head association of three to seven dimers. They were stable species capable of binding to, and subsequent release from, inside-out vesicles without degradation to tetramers or dimers. The data suggest that spectrin tetramers and medium-size oligomers coexist in the normal erythrocyte membrane as the primary native spectrin species.     

Simulations of the erythrocyte cytoskeleton at large deformation. I. Microscopic models.

Three variations of a polymer chain model for the human erythrocyte cytoskeleton are used in large deformation simulations of microscopic membrane patches. Each model satisfies an experimental observation that the contour length of the spectrin tetramers making up the erythrocyte cytoskeleton is roughly square root of 7 times the end-to-end distance of the tetramer in vivo. Up to modest stress, each brushy cytoskeletal network behaves, consistently, like a low-temperature, planar network of Hookean springs, with a model-dependent effective spring constant, keff, in the range of 20-40 kBT/s(o)2, where T is the temperature and s(o) is the force-free spring length. However, several features observed at large deformation distinguish these models from spring networks: 1) Network dimensions do not expand without bound in approaching a critical isotropic tension (square root of 3 keff) that is a characteristic limit of Hookean spring nets. 2) In surface compression, steric interactions among the chain elements prevent a network collapse that is otherwise observed in compression of planar triangulated networks of springs. 3) Under uniaxial surface tension, isotropy of the network disappears only as the network is stretched by more than 50% of its equilibrium dimensions. Also found are definitively non-Hookean regimes in the stress dependence of the elastic moduli. Lastly, determinations of elastic moduli from both fluctuations and stress/strain relations prove to be consistent, implying that consistency should be expected among experimental determinations of these quantities.     

A scanning electron microscopic study of the neural crest cell cytoskeleton after triton x-100 demebranation

Triton x-100, which solubilizes cell membranes, was used to expose the detergent resistant cytoskeletal systems of cultured day 4 (angulated) and a day 8 (STELLATE) NEURAL CREST cells. This procedure reveals a network of fibers which correlates structurally with the morphological state of neural crest cell differentiation.     

The effects of Korean propolis against foodborne pathogens and transmission electron microscopic examination

This study was performed to evaluate the effects of Korean propolis against foodborne pathogens and spores of Bacillus cereus and to investigate the antimicrobial activity against B. cereus structure by transmission electron microscopy (TEM). The antimicrobial effects of the Korean propolis were tested against foodborne pathogens including Gram-positive (B. cereus, Listeria monocytogenes and Staphylococcus aureus) and Gram-negative (Salmonella typhimurium, Escherichia coli and Pseudomonas fluorescence) bacteria by agar diffusion assay. Gram-positive bacteria were more sensitive than were Gram-negative bacteria. The vegetative cells of B. cereus were the most sensitive among the pathogens tested with minimum inhibitory concentration (MIC) of 0.036 mg/μl of propolis on agar medium. Based on MIC, sensitivity of vegetative cells of B. cereus and its spores was tested in a nutrient broth with different concentrations of propolis at 37°C. In liquid broth, treatment with 1.8 mg/ml propolis showed bactericidal effect against B. cereus. B. cereus vegetative cells exposed to 7.2mg/ml of propolis lost their viability within 20 min. Against spores of B. cereus, propolis inhibited germination of spores up to 30 hours, compared to control at higher concentration than vegetative cells yet acted sporostatically. The bactericidal and sporostatic action of propolis were dependent on the concentration of propolis used and treatment time. Electron microscopic investigation of propolis-treated B. cereus revealed substantial structural damage at the cellular level and irreversible cell membrane rupture at a number of locations with the apparent leakage of intracellular contents. The antimicrobial effect of propolis in this study suggests potential use of propolis in foods.     

Simulations of the erythrocyte cytoskeleton at large deformation. II. Micropipette aspiration.

Coarse-grained molecular models of the erythrocyte membrane's spectrin cytoskeleton are presented in Monte Carlo simulations of whole cells in micropipette aspiration. The nonlinear chain elasticity and sterics revealed in more microscopic cytoskeleton models (developed in a companion paper; Boey et al., 1998. Biophys. J. 75:1573-1583) are faithfully represented here by two- and three-body effective potentials. The number of degrees of freedom of the system are thereby reduced to a range that is computationally tractable. Three effective models for the triangulated cytoskeleton are developed: two models in which the cytoskeleton is stress-free and does or does not have internal attractive interactions, and a third model in which the cytoskeleton is prestressed in situ. These are employed in direct, finite-temperature simulations of erythrocyte deformation in a micropipette. All three models show reasonable agreement with aspiration measurements made on flaccid human erythrocytes, but the prestressed model alone yields optimal agreement with fluorescence imaging experiments. Ensemble-averaging of nonaxisymmetrical, deformed structures exhibiting anisotropic strain are thus shown to provide an answer to the basic question of how a triangulated mesh such as that of the red cell cytoskeleton deforms in experiment.