Localization of erythrocyte membrane antigens by immune electron microscopy

Torus (T) protein was prepared by differential centrifugation of hemoglobin-free membranes after freezing, thawing and dialysis against water. Ferritin-labeled antibody to T protein failed to agglutinate erythrocytes or to sensitize them to complement lysis. Anti-T bound specifically to the internal aspect of ghosts and to the exterior of inverted vesicles prepared from them. The findings lend further support to the supposition that native T protein exists only on the inner aspect of intact erythrocyte membranes; its possible relation to spectrin is uncertain. In contrast, ferritin-labeled antibody to purified erythrocyte glycoprotein (virus receptor substance, VRS) agglutinated erythrocytes, sensitized them to complement lysis and by its specific reactivity with surface antigen afforded a basis for the electron microscopic quantitation of receptor sites on plasma membranes examined in thin section as well as with freeze-etching.     

Sewage coliphages studied by electron microscopy.

Sewage was enriched with 35 Escherichia coli strains, and sediments of enrichment cultures were studied in the electron microscope. They contained up to 10 varieties of morphologically different particles. T-even-type phages predominated in 14 samples. Thirteen phages were enriched, representing the families Myoviridae (seven), Styloviridae (two), Podoviridae (three), and Microviridae (one). Twelve of these corresponded to known enterobacterial phage species, namely, 121, K19, FC3-9, O1, 9266, T2, 16-19, kappa, beta 4, N4, T7, and phi X174. Cubic RNA phages and filamentous phages were not detected. Types 121 and 9266 have previously been observed only in Romania and South Africa. Identification by morphology is usually simple. Our investigative technique is qualitative and will not detect all phages present. Most enrichment strains are polyvalent, and electron microscopy is always required for phage identification. In a general way, electron microscopy seems to be the method of choice for investigation of phage geography and ecology.     

Nucleoprotein-RNA orientation in the measles virus nucleocapsid by three-dimensional electron microscopy

Recombinant measles virus nucleoprotein-RNA (N-RNA) helices were analyzed by negative-stain electron microscopy. Three-dimensional reconstructions of trypsin-digested and intact nucleocapsids coupled to the docking of the atomic structure of the respiratory syncytial virus (RSV) N-RNA subunit into the electron microscopy density map support a model that places the RNA at the exterior of the helix and the disordered C-terminal domain toward the helix interior, and they suggest the position of the six nucleotides with respect to the measles N protomer.     

Internal structure of ovomacroglobulin studied by electron microscopy.

As a model for the molecular structure of proteins belonging to the alpha 2-macroglobulin family, ovomacroglobulin of reptilian origin was studied by electron microscopy in the original tetrameric form as well as in the dissociated forms into half- and quarter molecules. The following aspects of the molecular internal structure which had previously not been known for the homologous human alpha 2-macroglobulin or chicken ovomacroglobulin were revealed. First, the negatively stained tetrameric native protein gave an appearance of a collection of four semi-circular strings placed on the four corners of a molecule. They were connected to each other in the center of a molecule through a set of globular domains which formed a cross-figured subunit contact region. Second, two kinds of active half-molecules prepared either by the reduction of intersubunit disulfide bonds or by the disruption of noncovalent subunit interface had similarly elongated forms having semi-circular units on the two ends, indicating quasi-equivalent subunit arrangement in the two kinds of half-molecules. We thus concluded that the structure of native ovomacroglobulin can be represented by four circular strings each equipped with an extra domain to form the central intersubunit contact region. The results may also be adapted to the internal structure of human alpha 2-macroglobulin because it was sometimes possible to observe similar ring-like internal structure in the human protein.     

Constitutive plasma membrane targeting and microdomain localization of Dok5 studied by single-molecule microscopy

In the present study, single-molecule fluorescence microscopy was used to examine the characteristics of plasma membrane targeting and microdomain localization of enhanced yellow fluorescent protein (eYFP)-tagged wild-type Dok5 and its variants in living Chinese hamster ovary (CHO) cells. We found that Dok5 can target constitutively to the plasma membrane, and the PH domain is essential for this process. Furthermore, single-molecule trajectories analysis revealed that Dok5 can constitutively partition into microdomain on the plasma membrane. Finally, the potential mechanism of microdomain localization of Dok5 was discussed. This study provided insights into the characteristics of plasma membrane targeting and microdomain localization of Dok5 in living CHO cells.     

Spectrin-actin associations studied by electron microscopy of shadowed preparations

By shadowing specimens dried onto mica sheets we have obtained clear images of actin crosslinked by spectrin, an actin-binding protein found in erythrocytes. We conclude that spectrin dimers possess a single binding site for F actin. Tetramers formed by head-to-head association of two dimers possess two actin binding sites, one at each tail. Polymerizing G actin in the presence of spectrin tetramers or mixing preformed F actin with spectrin tetramer plus band 4.1 results in an extensively crosslinked network of actin filaments. When G actin is polymerized in the presence of spectrin at spectrin:actin mole ratios close to that present on the erythrocyte membrane, large amorphous protein networks are formed. These networks are clusters of spectrin around 25 nm diameter structures which may be actin protofilaments. These networks are similar to the cytoskeletal network seen after erythrocyte membranes are extracted with detergent, and may represent the first in vitro assembly of a cytoskeletal complex resembling that of the native cell both biochemically and structurally.     

The quatenary structure of Pseudomonas cytochrome oxidase studied by electron microscopy.

Pseudomonas cytochrome oxidase (EC 1.9.3.2) was studied by negative staining in the electron microscope. The best resolution was obtained with uranyl oxalate (pH 6.0) as negative stain. Electron micrographs confirm the idea of the dimeric structure of the enzyme. A rough model of cytochrome oxidase was constructed based on different projections of the molecule seen in the electron micrographs. In this model the subunits are identical and sterically equivalent.     

Membrane ultrastructure of alkaliphilic Bacillus species studied by rapid-freeze electron microscopy.

Cells of Bacillus firmus OF4 and Bacillus alcalophilus were examined by rapid-freeze freeze-fracture and freeze-substitution electron microscopy. No special vesicular structures linked to growth at alkaline pH were found, either within or associated with the cytoplasmic membrane. The cytoplasmic membranes of the alkaliphilic bacilli and the neutrophilic Bacillus subtilis BD99 were indistinguishable. Distinctive intramembrane particle rings, presumed to be flagellar structures on the basis of distribution and morphological characteristics, were found in all of these species. These observations indicate that the adaptations required to effect oxidative phosphorylation and flagellar rotation at extreme alkaline pH occur without gross morphological rearrangement.     

Morphological forms and viability of Campylobacter species studied by electron microscopy.

Electron microscopic studies of Campylobacter revealed that different morphological forms predominate at different parts of a colony. At the periphery, cells were almost all spirals, while in the center of the colony cells were mainly coccus shaped. Unusual ring-shaped cells, "donuts", were observed in the raised, peripheral region of the colony. Donut or ring forms have not previously been reported for Campylobacter organisms. Our data indicate that young or actively growing cells are mainly spiral shaped. Older cells undergo a degenerative change to coccoid forms. The donut shape appears to be an intermediate stage between spirals and cocci. Comparisons of plate counts of actively growing and inactive cells confirmed that coccoid cells are probably nonviable.     

Imaging of immunogold labelled antigens on capping thymocytes by confocal reflection contrast scanning optical microscopy

Confocal laser scanning optical microscopy (CLSM) in the reflection contrast mode has been used to image single 40 nm gold particles, and to study changes in the distribution of gold label associated with capping of the leukocyte sialoglycoprotein (LSGP) antigen on the surface of fixed rat thymocytes, labelled with the mouse monoclonal antibody W3/13 and a goat anti-mouse IgG immunogold conjugate. This imaging method has also been applied to live thymocytes labelled with gold-conjugated antibodies, to study the dynamics of the capping process.     

Tumour cell-tumour cell emperipolesis studied by transmission electron microscopy

During a transmission electron microscopic study of the JB-1 ascites tumour the rare phenomenon of tumour cell-tumour cell emperipolesis was observed. The ‘inner’ cell appeared in a vacuole of the ‘outer’ tumour cell.     

Structural insights into viral RNA capping and plasma membrane targeting by Chikungunya virus nonstructural protein 1

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Innervation of the guinea pig spleen studied by electron microscopy

The innervation of the guinea pig spleen was investigated by electron microscopy. Unmyelinated nerve fibers in the capsulotrabecular and arterial systems were found to contain large and small granular and small agranular synaptic vesicles in their terminals and are thought to be sympathetic adrenergic in nature. They influence the contraction of the smooth muscle cells by diffusion innervation in these systems. These nerve terminals were also scattered in both the red and the white pulp. Pulp nerves wrapped by Schwann cells were further enclosed by myofibroblastic reticular cells. This condition revealed that the pulp nerves pass through the connective-tissue spaces of the reticular fibers, which contain elastic fibers, collagenous fibrils, and lamina densa-like materials of the usual basement laminae. One of the target cells for the pulp nerves is considered to be the myofibroblastic reticular cell in the reticular meshwork. Neurotransmitter substances released from the naked adrenergic nerve terminals travel through the reticular fibers and may play a role, by both close association innervation and diffusion innervation, in the contraction of reticular cells to expose the reticular fibers. At the exposed sides, connective-tissue elements of the reticular fibers are bathed with blood plasma, and the included naked nerve terminals, devoid of Schwann cells but with basement laminae of these cells, face free cells at some distance or are in close association with free cells, especially lymphocytes, macrophages, and plasma cells. The close ultrastructural relationship between the naked adrenergic nerve terminals and immunocytes strongly suggests that there is an intimate relationship between the immune system and the sympathetic nervous system through both close association innervation and diffusion innervation. Thus splenic adrenergic nerves of the guinea pig may play a triple role in 1) contraction of smooth muscle cells to regulate blood flow in the organ, 2) induction of the exposure of reticular fibers by contraction of the reticular cells in order to form a close relationship of the nerve terminals with the immunocytes, and 3) subsequent neuroimmunomodulation of the immunocytes.     

Analysis of Borrelia burgdorferi membrane architecture by freeze-fracture electron microscopy.

Freeze-fracture electron microscopy was used to investigate the membrane architectures of high-passage Borrelia burgdorferi B31 and low- and high-passage isolates of B. burgdorferi N40. In all three organisms, fractures occurred almost exclusively through the outer membrane (OM), and the large majority of intramembranous particles were distributed randomly throughout the concave OM leaflet. The density of intramembranous particles in the concave OM leaflet of the high-passage N40 isolate was significantly greater than that in the corresponding leaflet of the low-passage N40 isolate. Also noted in the OMs of all three organisms were unusual structures, designated linear bodies, which typically were more or less perpendicular to the axis of the bacterium. A comparison of freeze-fractured B. burgdorferi and Treponema pallidum, the syphilis spirochete, revealed that the OM architectures of these two pathogens differed markedly. All large membrane blebs appeared to be bounded by a membrane identical to the OM of B. burgdorferi whole cells; in some blebs, the fracture plane also revealed a second bilayer closely resembling the B. burgdorferi cytoplasmic membrane. Aggregation of the lipoprotein immunogens outer surface protein A (OspA) and OspB on the bacterial surface by incubation of B. burgdorferi B31 with specific polyclonal antisera did not affect the distribution of OM particles, supporting the contention that lipoproteins do not form particles in freeze-fractured OMs. The expression of poorly immunogenic, surface-exposed proteins as virulence determinants may be part of the parasitic strategy used by B. burgdorferi to establish and maintain chronic infection in Lyme disease.