Assembly of chromatin fibers into metaphase chromosomes analyzed by transmission electron microscopy and scanning electron microscopy.

The higher-order assembly of the approximately 30 nm chromatin fibers into the characteristic morphology of HeLa mitotic chromosomes was investigated by electron microscopy. Transmission electron microscopy (TEM) of serial sections was applied to view the distribution of the DNA-histone-nonhistone fibers through the chromatid arms. Scanning electron microscopy (SEM) provided a complementary technique allowing the surface arrangement of the fibers to be observed. The approach with both procedures was to swell the chromosomes slightly, without extracting proteins, so that the densely-packed chromatin fibers were separated. The degree of expansion of the chromosomes was controlled by adjusting the concentration of divalent cations (Mg2+). With TEM, individual fibers could be resolved by decreasing the Mg2+ concentration to 1.0-1.5 mM. The predominant mode of fiber organization was seen to be radial for both longitudinal and transverse sections. Using SEM, surface protuberances with an average diameter of 69 nm became visible after the Mg2+ concentration was reduced to 1.5 mM. The knobby surface appearance was a variable feature, because the average diameter decreased when the divalent cation concentration was further reduced. The surface projections appear to represent the peripheral tips of radial chromatin loops. These TEM and SEM observations support a "radial loop" model for the organization of the chromatin fibers in metaphase chromosomes.     

Stereo electron microscopy of the 25-nm chromatin fibers in isolated nuclei

The morphological effects of epidermal growth factor (EGF) on human carcinoma cells A-431 have been examined by scanning electron microscopy. These flat polygonal cells normally exhibit only small membrane folds, but show extensive ruffling and extension of filopodia within 5 min of exposure to EGF at 37 degrees C. This ruffling activity is transient, subsiding within another 5--15 min, but several other changes in surface morphology follow. Within the first hour of exposure to the hormone, the cell surface becomes exceedingly smooth and the nuclei seem to protrude above the plane of the otherwise thin monolayer, giving the cells a "fried egg" appearance. Cells at the edges of colonies gradually retract from the substrate, leading to reorganization, by 12 h, of the monolayer into multilayered colonies. EGF thus induces both rapid and long-term alterations in the morphology of these epidermoid cells.     

Local cell membrane deformations due to receptor-ligand bonding as seen by reflection microscopy

Understanding surface receptor clustering and redistribution processes at the cell-matrix contact zone requires detailed knowledge of the spatial integration of these molecules in the architecture of this complex interface. Here we present and discuss critically a procedure to extract such information combining reflection contrast microscopy (RCM) and reflection interference microscopy (RIM). As model system, we used living human umbilical vein endothelial cells (HUVEC) adhering to laminin-coated surfaces and investigated the distribution of the alpha2beta1 (CD29/CD49b) integrin at the contact zone of these cells. First, we applied freeze-fracture electron microscopy to gain information on microscopic details of the alpha2beta1 distribution at the contact zone. Next, we visualized and analyzed the overall lateral distribution of the integrins applying RCM using immunogold-labeling with 10 nm labels and a special silver enhancement technique. We found that RCM can be used to determine the lateral position of the marked receptor molecules to an accuracy of about 100-200 nm, instead of large morphological changes at the contact zone during silver enhancement. Finally, we combined RCM with RIM and analyzed the interference pattern of the contact zone around the label positions. Thus, we were able to detect changes of the average shape of the cell membrane due to receptor-ligand bonding of a size down to the resolution of the techniques.     

Ultrastructure and nuclear architecture of telomeric chromatin revealed by correlative light and electron microscopy

Telomeres, the ends of linear chromosomes, are composed of repetitive DNA sequences, histones and a protein complex called shelterin. How DNA is packaged at telomeres is an outstanding question in the field with significant implications for human health and disease. Here, we studied the architecture of telomeres and their spatial association with other chromatin domains in different cell types using correlative light and electron microscopy. To this end, the shelterin protein TRF1 or TRF2 was fused in tandem to eGFP and the peroxidase APEX2, which provided a selective and electron-dense label to interrogate telomere organization by transmission electron microscopy, electron tomography and scanning electron microscopy. Together, our work reveals, for the first time, ultrastructural insight into telomere architecture. We show that telomeres are composed of a dense and highly compacted mesh of chromatin fibres. In addition, we identify marked differences in telomere size, shape and chromatin compaction between cancer and non-cancer cells and show that telomeres are in direct contact with other heterochromatin regions. Our work resolves the internal architecture of telomeres with unprecedented resolution and advances our understanding of how telomeres are organized in situ.     

Protoperidinium (Dinophyceae) from Greece as seen by scanning electron microscopy

The marine dinoflagellates Protoperidinium ventricum (Abé) Balech, P. diaholus (Cleve) Balech, P. tristylum (Stein) Balech and P. divergens (Ehrenberg) Balech, collected from neritic plankton of the gulfs of Saronikos and Korinthiakos, Greece, have been examined by SEM. Morphological characteristics of their thecae are described and evaluated. Results are compared with patterns established by earlier SEM studies on related taxa.     

Structure of human platelet membrane glycoproteins IIb and IIIa as determined by electron microscopy

The glycoprotein (GP) IIb-IIIa complex was isolated from human platelet membranes and examined for glycoprotein stoichiometry and morphology. To determine the ratio of glycoproteins in the complex, the isolated glycoproteins were solubilized with sodium dodecyl sulfate and separated by high-performance liquid chromatography. Quantitative amino acid analysis of individual glycoproteins showed that the ratio of GP IIb to GP IIIa in the Ca2+-dependent complex was 0.93:1. Morphology was determined by electron microscopy of rotary-shadowed and negatively stained specimens. Individual complexes consisted of two domains: an oblong head of approximately 8 X 10 nm with two rodlike tails extending approximately 14-17 nm from one side of the head. Treatment of the isolated complex with EDTA resulted in the appearance of a mixture of oblong and filamentous structures, which could be separated by a sucrose gradient sedimentation in Triton X-100. As seen by rotary and unidirectional shadowing, GP IIb was a compact structure, approximately 8 X 10 nm in size. Isolated GP IIIa was more heterogeneous but was most often observed in an elongated form, varying in length from 20 to 30 nm and in width from 2 to 3 nm. By comparing these structures to that of the heterodimer complex, it was determined that the oblong domain was GP IIb and the rodlike tails were GP IIIa. Each milligram of isolated GP IIb-IIIa complex bound 0.30 mg of [3H]Triton X-100, indicating that the glycoprotein complex contained limited hydrophobic domains. Upon removal of detergent, GP IIb-IIIa complexes formed aggregates that sedimented in sucrose gradients as a diffuse peak ranging from 14 to 32 s. Examination of these aggregates by electron microscopy showed that they were composed of clusters or "rosettes" of 2 to 20 or more of the GP IIb-IIIa complexes. The orientation of these rosettes was such that the tails were joined in the center, with the head portions directed away from the interacting tails. It thus appears that the primary hydrophobic domains of the GP IIb-IIIa complex exist at the tips of the GP IIIa tails. Because the GP IIb-IIIa complex is an intrinsic membrane glycoprotein, these findings indicate a potential membrane attachment site for the GP IIb-IIIa complexes.     

Healing of microvascular arterial anastomoses, as seen on corrosion casts by scanning electron microscopy.

We made 44 microsurgical anastomoses in the femoral artery in rats. The healing was studied from one day to 4 months, by making an acrylic cast of the lumen of the femoral artery and the surrounding vessels, and examining the cast by scanning electron microscopy. The method proved to be easy and relatively quick, and it limited the possibility of artifacts. The high depth of focus permitted simultaneous study of the main lumen and of the perivascular structures after microvascular anastomoses.     

Special features of phosphatidylcholine vesicles as seen in cryo-transmission electron microscopy

Vesicles of egg yolk phosphatidylcholine (EYPC) were studied by cryo-transmission electron microscopy. The electron micrographs indicate that, despite the rapidity of cooling, membrane undulations are flattened and some vesicles change their shapes before the samples freeze. These artefacts are attributed to the action of the lateral tension that results from the membrane area contraction associated with the temperature drop. Other micrographs represent grainy membranes and angular vesicles. We regard them as the first direct evidence for the superstructure and optically invisible roughness which were recently postulated for these membranes.     

Helicoids in the T system and striations of frog skeletal muscle fibers seen by high voltage electron microscopy.

Reconstruction from thick serial transverse slices of frog skeletal muscle fibers stained with peroxidase and examined by high-voltage electron microscopy has revealed that the T system networks at successive sarcomeres are connected together in a helicoidal fashion. From zero to eight helicoids have been found in each of a group of 21 fibers reconstructed in cross section. Helicoids can have either right- or left-handed screw senses, and both senses can be found in one fiber cross section. Because the T system maintains a relatively precise alignment with the myofibrillar striations, it follows that the striations must also have a helicoidal arrangement. This has been found before, but has not been widely accepted in recent times. The presence of helicoids in the bands and membrane networks is not thought per se to alter very much our thinking about excitation and contraction mechanisms in skeletal muscle fibers.     

Characterization of restriction nuclease prepared chromatin by electron microscopy

Chromatin was solubilized from rat liver nuclei by digestion with the restriction nuclease EcoRI or HaeIII in the presence or absence of EDTA and sodium chloride. The samples were investigated by electron microscopy after positive and negative staining with uranyl acetate under a number of conditions. Depending on the salt concentration during solubilization the chromatin appeared as beads on the string or in more compact form. Solenoid- and superbead-like structures were seen as had been reported for chromatin solubilized with micrococcal nuclease.     

Ornamentation of thecal plates in Protoperidinium (Dinophyceae) as seen by scanning electron microscopy

The thecae of over thirty species of Protoperidinium from aroundthe British Isles have been examined by SEM and the varioustypes of ornamentation described. The simplest type consistsof smooth plates in which the trichocyst pores have an evenor irregularly thickened rim. In one species the pores are situatedon mounds. In another variation the plates are covered withminute pimples. Many species have plates which are thickenedby a surface reticulum and this may be made up of lines of pimples,of irregularly arranged ridges or bars which protrude to formsmall spines. In one species the ridges always surround trichocystpores and in another there are prominent spines which bear noobvious relationship with the pores. It is concluded that thecalornamentation can provide useful taxonomic characters withinthis genus.     

Histone-histone proximity in chromatin as seen by imidoester cross-linking

After treatment of purified chromatin with dimethyl adipimidate (a reversible crosslinking reagent) a number of histone oligomers could be isolated in the soluble form from the chromatin. It was shown that all five histones take part in the dimethyl adipimidate-induced oligomer formation. Only a few kinds of histone oligomers (with unknown composition) could be isolated in the soluble form after treatment of chromatic with formaldehyde.