[Modification of the method of metal shadowing, applied to the study of histological sections examined under the optic microscope].
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Ultrastructure and immunocytochemistry of the isolated human erythrocyte membrane skeleton.
Isolated skeletons from human erythrocyte ghosts were studied using immunogold labeling; negative staining; and quick-freeze, deep-etch, rotary replication with Pt/C (QFDERR). Isolated skeletons visualized by QFDERR were similar to the negatively stained skeletons in that the proteins spectrin, actin, and ankyrin could be easily distinguished. However, the quick-frozen skeletons had two fewer filaments (4.2 +/- 0.7) at an actin junction. Immunogold labeling of skeletons with site-specific spectrin antibodies not only confirmed the designation of these filaments as spectrin molecules, but indicated that about 30% of spectrin filaments form non-actin junctions consistent with the hexameric organization of these filaments. Many of the filaments displayed a striking banding pattern indicative of underlying substructure. Isolated skeletons prepared by QFDERR also showed evidence of laterally associated spectrin filaments. These associations, as well as many hexamer junctions, are lost during negative staining. Negative staining also apparently caused approximately 21% of the spectrin filaments to separate into their monomeric subunits. These results indicate that the surface tension imposed during negative staining of isolated skeletons can cause a loss of interactions normally present in the intact membrane skeleton.
Erythrocyte Membrane↗
Visualization of the unwinding of long DNA chains by the herpes simplex virus type 1 UL9 protein and ICP8.
UL9 protein and ICP8 encoded by the herpes simplex virus type 1 (HSV-1) were shown to catalyze a highly active, non-origin-dependent unwinding of DNA. UL9 protein, the HSV-1 origin binding protein, as a modest helicase activity that is greatly stimulated by the HSV-1 single strand (ss) binding protein, ICP8. Here, electron microscopy has been applied to examine the mechanics of this reaction. Negative staining of the proteins revealed particles consisting primarily of ICP8 monomers and UL9 protein dimers. When the binding of UL9 protein to double strand (ds) DNA containing ss tails was examined by shadowcasting methods, UL9 protein was seen bound to the ss tails or ss/ds junctions; addition of ATP led to its appearance internally along the ds segment. When UL9 protein and ICP8 were incubated together with the tailed dsDNA in the presence of ATP, a highly ordered unwinding of the DNA was observed by negative staining that appeared to progress through four distinct stages: (1) binding of ICP8 to the ss tail and progressive coverage of the ds portion by UL9 protein; (2) formation of highly condensed regular filaments; (3) relaxation of the condensed structures into coiled-coils; and (4) unwinding of the coils and release of ICP8-covered linear ssDNAs. This process represents a mechanism of unwinding that is very different from ones that proceed by a progressive unwinding at Y-shaped forks that move along the DNA.
Adenosine Triphosphate↗
An atomic model of crystalline actin tubes: combining electron microscopy with X-ray crystallography.
The packing of the G-actin monomers within crystalline actin tubes was investigated at atomic detail. To achieve this, we have chosen an integrated structural approach which combines intermediate resolution electron microscopy based 3-D reconstruction and surface metal shadowing of crystalline actin tubes with atomic resolution X-ray data of the G-actin monomer. Distinct from the parallel, half-staggered packing of the actin subunits within F-actin filaments, the arrangement of actin monomers within the crystalline tubes involves antiparallel packing into dimers with p2 symmetry. Within the crystalline tubes, the actin monomers are oriented so that the filament axis runs parallel with the sheet plane and the intersubunit contacts in this direction are similar to those existing along the two long-pitch helical strands of the F-actin filament. The other intersubunit contacts within the crystalline tubes are not found in the actin filament. The ability of actin to form a variety of polymorphic oligomers is still not fully understood, and the functional implications of this variability have yet to be deciphered. Regularly packed actin assemblies such as sheets, tubes or ribbons may ultimately yield structural relationships to in vivo relevant actin oligomers such as, for example, the "lower dimer".
Actins↗
The aquaporin sidedness revisited.
Aquaporins are transmembrane water channel proteins, which play important functions in the osmoregulation and water balance of micro-organisms, plants, and animal tissues. All aquaporins studied to date are thought to be tetrameric assemblies of four subunits each containing its own aqueous pore. Moreover, the subunits contain an internal sequence repeat forming two obversely symmetric hemichannels predicted to resemble an hour-glass. This unique arrangement of two highly related protein domains oriented at 180 degrees to each other poses a significant challenge in the determination of sidedness. Aquaporin Z (AqpZ) from Escherichia coli was reconstituted into highly ordered two-dimensional crystals. They were freeze-dried and metal-shadowed to establish the relationship between surface structure and underlying protein density by electron microscopy. The shadowing of some surfaces was prevented by protruding aggregates. Thus, images collected from freeze-dried crystals that exhibited both metal-coated and uncoated regions allowed surface relief reconstructions and projection maps to be obtained from the same crystal. Cross-correlation peak searches along lattices crossing metal-coated and uncoated regions allowed an unambiguous alignment of the surface reliefs to the underlying density maps. AqpZ topographs previously determined by AFM could then be aligned with projection maps of AqpZ, and finally with human erythrocyte aquaporin-1 (AQP1). Thereby features of the AqpZ topography could be interpreted by direct comparison to the 6 A three-dimensional structure of AQP1. We conclude that the sidedness we originally proposed for aquaporin density maps was inverted.
Aquaporin 1↗
Electron microscopic observation and single-stranded DNA binding activity of the Mcm4,6,7 complex.
Mcm2-7 proteins that play an essential role in eukaryotic DNA replication contain DNA-dependent ATPase motifs in a central domain that, from yeast to mammals, is highly conserved. Our group has reported that a DNA helicase activity is associated with a 600 kDa human Mcm4, 6 and 7 complex. The structure of the Mcm4,6,7 complex was visualized by electron microscopy after negative staining with uranyl acetate. The complex contained toroidal forms with a central channel and also contained structures with a slit. Gel-shift analysis indicated that the level of affinity of the Mcm4,6,7 complex for single-stranded DNA was comparable to that of SV40 T antigen, although the Mcm4,6,7 complex required longer single-stranded DNA for the binding than did SV40 T antigen. The nucleoprotein complexes of Mcm4,6,7 and single-stranded DNA were visualized as beads in a queue or beads on string-like structures. The formation of these nucleoprotein complexes was erased by Mcm2 that is a potential inhibitor of the Mcm4,6,7 helicase. We also found that the DNA helicase activity of Mcm4,6,7 complex was inhibited by the binding of Mcm3,5 complex. These results support the notion that the Mcm4,6,7 complex functions as a DNA helicase and the formation of 600 kDa complex is essential for the activity.
Animals↗
Structural conversion between open and closed forms of radixin: low-angle shadowing electron microscopy.
The function of ERM (ezrin/radixin/moesin) proteins as general cross-linkers between actin filaments and plasma membranes is regulated downstream of Rho, through the transition between active and inactive forms. To directly examine the conformational change between the active and inactive forms of ERM proteins, we applied low-angle rotary-shadowing electron microscopy to the radixin molecules, wild-type, T564A-non-phosphorylated-type, and T564E-phosphorylated-type, since most of the active forms are reportedly stabilized in cells by the C-terminal threonine phosphorylation. As a result, the T564A- and wild-type radixin molecules yielded the globular closed forms, approximately 8-14 nm in diameter, with some striations on their surfaces. In contrast, the T564E-radixin molecules tended to take elongated open forms, in which two globular structures measuring approximately 8 nm and approximately 5 nm in diameter were associated with both ends of the filamentous structures. The filamentous structure took either a approximately 20-25 nm-long straight course or a folded course. Taken together with the biochemical and the crystal structural results obtained to date, the closed and open forms represent the inactive and active forms of radixin as cross-linkers between actin filaments and plasma membranes.
Amino Acid Substitution↗
Improved procedures for electron microscopic visualization of the cytoskeleton of cultured cells.
We have developed an improved electron microscopic procedure appropriate for correlative light and electron microscopy of the cytoskeleton. The procedure is based on detergent extraction, chemical fixation, critical point drying, and platinum/carbon coating of cultured cells and the improvements consist of modifications which are minor individually but collectively of substantial impact. They are: inclusion of polyethylene glycol into the extraction medium; cell lysis at room temperature; fixation by sequential application of glutaraldehyde, tannic acid, and uranyl acetate; horizontal position of specimens during dehydration and drying; and uranyl acetate treatment during dehydration. As a result, we have obtained a greatly improved quality of electron microscopic images together with a high consistency of results. Long and straight actin filaments were clearly seen in stress fibers and newly formed lamellipodia. Their polarity was distinctly revealed by decoration with myosin subfragment 1. Depletion of actin from cytoskeletons by gelsolin treatment allowed for better visualization of myosin, intermediate filaments, and microtubules. Intermediate filaments exposed by this treatment exhibited numerous side projections in a hitherto unreported millipede-like appearance. The suggested procedure was compatible with immunogold labeling as demonstrated with an antibody to tubulin. Correlative light and electron microscopy of cells microinjected with a fluorescent derivative of myosin II was reliable and efficient, producing a close resemblance between the two kinds of images.
3T3 Cells↗
Rotary and unidirectional metal shadowing of VAT: localization of the substrate-binding domain.
AAA-ATPases have important roles in manifold cellular processes. VAT (valosine-containing protein-like ATPase of Thermoplasma acidophilum), a hexameric archaeal member of this family, has the tripartite domain structure N-D1-D2 that is characteristic of many members of this family. N, the N-terminal domain of 20.5 kDa, has been implicated in substrate binding. We have applied rotary and unidirectional shadowing to VAT and an N-terminally deleted mutant, VAT(Delta N), in order to map the location of this domain. For the analysis of data derived from unidirectionally shadowed samples we used a new approach combining eigenvector analysis with surface relief reconstruction. Averages of rotary shadowed particles as well as relief reconstructions map the N-terminal domains to the periphery of the hexameric complex and reveal their bipartite structure. Thus, this method appears to be well suited to study the conformational changes that occur during the functional cycle of the protein.
Adenosine Triphosphatases↗
Three-dimensional study of the cytoskeleton in macrophages and multinucleate giant cells by quick-freezing and deep-etching method.
The three-dimensional ultrastructure of multinucleate giant cells in subcutaneous granulomas was compared with those of peritoneal macrophages using a quick-freezing and deep-etching method. Subcutaneous granulomas were induced by implanting plastic coverslips in the dorsal subcutaneous tissue of rats. The quick-freezing and deep-etching replicas were prepared from the cells attached to the coverslips. Dense networks of actin filaments were distributed along all peripheral aspects (beneath the plasma membrane, and on free and coverslip-attached surfaces) of the multinucleate giant cells. On the coverslip-attached surface, numerous clathrin-coated pits and vesicles occurred between the actin filaments. In these cells, intermediate filaments, but not actin filaments, were the predominant cytoskeletal components in perinuclear regions and were attached to the cell nucleus, mitochondria and other vesicular cell organelles. A similar distribution of cytoskeletal components was observed in the mononuclear macrophages of the granulomas and the peritoneal macrophages. These results show that the cytoskeletal organization varies in different regions of the cytoplasm of multinucleate giant cells, while the characteristic cytoskeletal arrangement, resembling that of mononuclear macrophages, is maintained.
Actins↗
Cuticular Architecture of Hassalstrongylus epsilon (Nematoda: Trichostrongyloidea).
Hassalstrongylus epsilon is a small nematode, whose adult forms are found among the intestinal microvilli of the water rat Nectomys squamipes, Brants 1827 (Rodentia: Muridae). The external appearance of the cuticle, which presents transversal striations and longitudinal ridges, is described using scanning electron microscopy. Transmission electron microscopy of thin sections and replicas of quick-frozen, freeze-fractured, deep-etched and rotatory shadowed samples showed the presence in the cuticle of struts that arise from the fluid median layer, extending outward to the epicuticle. The cuticle showed the presence of five layers: epicuticle, cortical, fibril-rich, fluid median and fibrous. The cuticle layers were made of an assemblage of fibers that create compartments, which were larger in the fluid region than in the fibril-rich median layer.
Animals↗
2D crystallization: from art to science.
The techniques as well as the principles of the 2D crystallization of membrane and water-soluble proteins for electron crystallography are reviewed. First, the biophysics of the interactions between proteins, lipids and detergents is surveyed. Second, crystallization of membrane proteins in situ and by reconstitution methods is discussed, and the various factors involved are addressed. Third, we elaborate on the 2D crystallization of water-soluble proteins, both in solution and at interfaces, such as lipid monolayers, mica, carbon film or mercury surfaces. Finally, techniques and instrumentations that are required for 2D crystallization are described.
Aluminum Silicates↗
Structure of functional single AQP0 channels in phospholipid membranes.
Aquaporin-0 (AQP0) is the most prevalent intrinsic protein in the plasma membrane of lens fiber cells where it functions as a water selective channel and also participates in fiber-fiber adhesion. We report the 3D envelope of purified AQP0 reconstituted with random orientation in phospholipid bilayers as single particles. The envelope was obtained by combining freeze-fracture, shadowing and random conical tilt electron microscopy followed by single particle image processing. Two-dimensional analysis of 2547 untilted images produced eight class averages exhibiting "square" and "octagonal" shapes with a continuum of variation. We reconstructed in 3D five class averages that best described the data set. The reconstructions ("molds") appeared as metal cups exhibiting external and internal surfaces. We used the internal surface of the mold to calculate the "imprints" that represent the AQP0 particles protruding from the hydrophobic core of the phospholipid bilayer. The complete envelope of the channel, formed by joining the square and octagonal imprints, described accurately the size, shape, oligomeric state, orientation, and molecular weight of the AQP0 channel inserted in the phospholipid bilayer. Rigid body docking of the atomic model of the aquaporin-1 (AQP1) tetramer showed that the freeze-fracture envelope accounted for the conserved transmembrane domain (approximately 73% similarity between AQP0 and AQP1) but not for the amino and carboxyl termini. We suggest that the discrepancy might reflect differences in the location of the amino and carboxyl termini in the crystal and in the phospholipid bilayer.
Animals↗
Efficacy of argon laser irradiation in removing intracanal debris.
Endodontic instrumentation, irrigation, or intracanal medication are unable to totally remove intracanal debris. The purpose of this study was to evaluate the efficacy of argon laser irradiation in removing debris from the root canal system. After cleaning and shaping, 12 single rooted teeth were randomly divided into two groups of six teeth each. The first group, which served as the control, was not lased. In the second group, a 300 microns argon laser fiber optic was introduced into the root canal of each tooth, to its working length. Fifteen pulses of 100 msec each with an energy of 2 watts were delivered for 5 seconds at the apex. The lasing procedure was repeated at 1 mm intervals along the root canal, and the fiber tip was retrieved from the apex to the orifice. Computerized scanning electron microscopy analysis revealed that the amount of debris in the lased group was significantly lower than that in the control (p = 0.0001). On the basis of these results, it appeared that intracanal argon laser irradiation was an efficient mean of removing intracanal debris.
Argon↗
Proteodermatan and proteokeratan sulfate (decorin, lumican/fibromodulin) proteins are horseshoe shaped. Implications for their interactions with collagen.
The small proteoglycans proteodermatan and proteokeratan sulfates organize collagen fibrils in extracellular matrix [Scott, J. E. (1992) FASEB J. 6, 2639-2645], thus helping to maintain tissue shape. Their interaction with fibrils is probably via the protein. They have been examined by rotary shadowing-electron microscopy, which showed that these leucine-rich-repeat proteins are horseshoe shaped. Morphometry and comparison with polypeptide sequences suggest ways in which decorin could interact with tissue collagen fibrils. It is proposed that decorin is a bidentate ligand attached to two parallel neighboring collagen molecules in the fibril, helping to stabilize fibrils and orient fibrillogenesis.
Amino Acid Sequence↗
Structure of the gamma-tubulin ring complex: a template for microtubule nucleation.
The gamma-tubulin ring complex (gammaTuRC) is a protein complex of relative molecular mass approximately 2.2 x 10(6) that nucleates microtubules at the centrosome. Here we use electron-microscopic tomography and metal shadowing to examine the structure of isolated Drosophila gammaTuRCs and the ends of microtubules nucleated by gammaTuRCs and by centrosomes. We show that the gammaTuRC is a lockwasher-like structure made up of repeating subunits, topped asymmetrically with a cap. A similar capped ring is also visible at one end of microtubules grown from isolated gammaTuRCs and from centrosomes. Antibodies against gamma-tubulin label microtubule ends, but not walls, in centrosomes. These data are consistent with a template-mediated mechanism for microtubule nucleation by the gammaTuRC.
Animals↗
Beta-helix model for the filamentous haemagglutinin adhesin of Bordetella pertussis and related bacterial secretory proteins.
Bordetella pertussis establishes infection by attaching to epithelial cells of the respiratory tract. One of its adhesins is filamentous haemagglutinin (FHA), a 500-A-long secreted protein that is rich in beta-structure and contains two regions, R1 and R2, of tandem 19-residue repeats. Two models have been proposed in which the central shaft is (i) a hairpin made up of a pairing of two long antiparallel beta-sheets; or (ii) a beta-helix in which the polypeptide chain is coiled to form three long parallel beta-sheets. We have analysed a truncated variant of FHA by electron microscopy (negative staining, shadowing and scanning transmission electron microscopy of unstained specimens): these observations support the latter model. Further support comes from detailed sequence analysis and molecular modelling studies. We applied a profile search method to the sequences adjacent to and between R1 and R2 and found additional "covert" copies of the same motifs that may be recognized in overt form in the R1 and R2 sequence repeats. Their total number is sufficient to support the tenet of the beta-helix model that the shaft domain--a 350 A rod--should consist of a continuous run of these motifs, apart from loop inserts. The N-terminus, which does not contain such repeats, was found to be weakly homologous to cyclodextrin transferase, a protein of known immunoglobulin-like structure. Drawing on crystal structures of known beta-helical proteins, we developed structural models of the coil motifs putatively formed by the R1 and R2 repeats. Finally, we applied the same profile search method to the sequence database and found several other proteins--all large secreted proteins of bacterial provenance--that have similar repeats and probably also similar structures.
Adhesins, Bacterial↗
Crosslinking of fibrinogen and fibronectin by free radicals: a possible initial step in adhesion formation in osteoarthritis of the temporomandibular joint.
PURPOSE: Adhesion formation in osteoarthritis (OA) of the temporomandibular joint (TMJ) typically results in a sustained limitation of joint movement. We propose the hypothesis that free-radical-mediated crosslinking of proteins underlies this adhesion formation in affected joints. Free radicals may cause oxidative modification of proteins, creating an opportunity for the formation of intramolecular and intermolecular crosslinks via covalent bonds. This may stabilize protein aggregates, rendering them more resistant to degradation. In this study, the free-radical-mediated crosslinking of model proteins (fibrinogen and fibronectin) was investigated to test our hypothesis that free radicals contribute to adhesion formation via this mechanism in OA of the TMJ. MATERIALS AND METHODS: Physiological clot formation of fibrinogen by thrombin and free-radical-induced crosslinking of fibrinogen and of fibronectin were analyzed using spectrophotometric turbidity measurements, light-scattering techniques, polyacrylamide gel electrophoresis (PAGE), and rotary shadowing. RESULTS: Fibrinogen was shown to aggregate after free radical treatment, as detected using turbidity measurements and light-scattering techniques. Using PAGE, fibrinogen as well as fibronectin was shown to degrade under low oxidative stress. Under high oxidative stress, however, fragments from both proteins were found to be covalently crosslinked, resulting in high-molecular-weight protein aggregates. The aggregation was shown to be at random with rotary shadowing. CONCLUSION: The study shows that high oxidative stress contributes to the formation of crosslinked proteins that may serve as an initial scaffolding for the development of adhesions frequently seen in OA of the TMJ.
Cross-Linking Reagents↗