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Rapid freeze-freeze substitution after glutaraldehyde fixation (CF-FS method) obtained the natural and fine structures of macrophages and engulfed yeast cells. Culturing macrophages on single hole molybdenum grids placed in culture dishes made possible the rapid freezing of cells by the 'open sandwich method'. This method may be convenient when rapid-freezing cannot be performed immediately, or when a rapid-freezing device is not available in the lab.

The unique long-neck yeast Fellomyces fuzhouensis has F-actin cables and cortical patches. Here, we describe a new F-actin structure present in fungi, a perinuclear F-actin collar ring around the cell nucleus. This F-actin structure can be visualized by fluorescent microscopic imaging of rhodamine-phalloidin-stained F-actin in cells treated with the mitotic drug isopropyl N-(3-chlorophenyl) carbamate or the microtubule inhibitor thiabendazol or when cells were grown in cut dried radish medium or yeast extract pepton dextrose (YEPD) medium. In contrast, these structures were absent in cells treated with Latrunculin A. The hypothetical functions of the F-actin ring are discussed.

Temperature-sensitive actin mutant of Saccharomyces cerevisiae act1-1 was studied at a permissive temperature of 23 C by light, fluorescent and electron microscopy to elucidate the roles of actin cytoskeleton in the cycling eukaryotic cells. Mutant cells that grew slowly at the permissive temperature showed aberrations in the cytoskeleton and cell cycle. Mutant cells contained aberrant ‘faint actin cables,’ that failed in directing of mitochondria, vacuoles and secretory vesicles to the bud and the stray vesicles delivered their content to the mother wall instead of the bud. Bud growth was delayed. Spindle pole bodies and cytoplasmic microtubules did not direct to the bud, and nucleus failed to migrate to the bud. Repeated nuclear divisions produced multinucleated cells, indicating continued cycling of actin mutant cells that failed in the morphogenetic checkpoint, the spindle position checkpoint and cytokinesis. Thus, a single actin mutation appears to indicate uncoupling in space and time of the ‘actin cytoskeleton-dependent cytoplasmic pathway of bud development and organelle positioning and inheritance’ from the ‘microtubule- dependent nuclear division pathway’ in a budding yeast cell cycle.

The spindle pole body (SPB) in the interphase cell of the pathogenic yeast Cryptococcus neoformans was studied in detail by freeze-substitution and serial ultrathin sectioning electron microscopy. The SPB was located on the outer nuclear envelope and appeared either dumbbell- or bar shaped. The dumbbell-shaped SPBs were 228-365 nm long with amorphous spheres on each end, each sphere being 78-157 nm in diameter. The bar-shaped SPBs were 103-260 nm long and 32-113 nm thick. They consisted of filamentous materials. The dumbbell-shaped SPBs were more frequent (61%) than the bar-shaped SPBs. The bar-shaped SPBs may be regarded as dumbbell-shaped SPBs whose spherical parts became sufficiently small. There seemed to be no relationship between the SPB shape and the cell cycle stage of G1-G2, since both types of SPB appeared not only in unbudded cells but also in budded cells and their appearance seems to be random. It is not clear at present whether morphological changes between dumbbell- and bar shapes have any physiological function. The SPB tended to be localized away from the nucleolus (141 degrees +/- 44 degrees), but localized randomly to the bud (97 degrees +/- 50 degrees). The present study highlights the necessity of observing a large number of micrographs in three dimensions to describe accurately the ultrastructure of the SPB in yeast.

A smart and efficient method for freeze substitution and serial sectioning of yeast cells is described. Yeast cells were placed in a single layer between two copper disks, rapidly frozen, freeze substituted and embedded in an epoxy resin. The cell layer was re-embedded by the same resin, the surface trimmed leaving 1 mum above the cell layer, and serially sectioned. The sections were collected on the two-slit grids and placed on a Formvar film mounted to cover the holes of an aluminum supporting rack. The grids were removed from the rack, stained together using a silicon tube and observed in a transmission electron microscope. The images of yeast cells observed were clear and natural, and would be useful for a detailed 3D structural analysis such as structome.

The cell structure has been studied using light and electron microscopies for centuries, and it is assumed that the whole structure is clarified by now. Little quantitative and three-dimensional analysis of cell structure, however, has been undertaken. We have coined a new word, 'structome', by combining 'structure' and '-ome', and defined it as the 'quantitative and three-dimensional structural information of a whole cell at the electron microscopic level'. In the present study, we performed structome analysis of Saccharomyces cerevisiae, one of the most widely researched biological materials, by using freeze-substitution and serial ultrathin-sectioning electron microscopy. Our analysis revealed that there were one to three mitochondria, ~220 000 ribosomes in a cell, and 13-28 endoplasmic reticula/Golgi apparatus which do not form networks in the cytoplasm in the G1 phase. Nucleus occupied ~10.5% of the cell volume; cell wall occupied ~17%; vacuole occupied ~5.8%; cytoplasm occupied ~64%; and mitochondria occupied only ~1.7% in the G1 phase. Structome analysis of cells would form a base for the post-genome research.

A method was proposed for improving preservation of ultrastructures of deep-sea microorganisms by using rapid-freeze freeze-substitution after glutaraldehyde fixation. This method produced clear high-resolution images of cells appearing in their natural state, close to the quality of images obtained by rapidly freezing freeze-substituted specimens of living cells. The method may be useful for observing any microorganism when rapid freezing of living samples is difficult and only glutaraldehyde fixation can be carried out.

Protoplast regeneration of a wild-type and two mutant strains of Candida glabrata defective in CHS3 homologues encoding class IV chitin synthase in Saccharomyces cerevisiae was examined by scanning and negative-staining electron microscopy. In the wild-type strain, small particles and short filaments appeared on the protoplast surface at 10 min, filamentous materials covered the entire surface of the protoplast at 1 h, granular materials started filling interspaces of filamentous materials at 2 h and regeneration was completed at 6 h. The filamentous materials consisted of microfibrils of various widths ranging from ≤5 to 40 nm, and composed of β-glucan. Protoplasts of the two chitin synthase mutant strains of Δchs3A and Δchs3B completed regeneration essentially by the same process as wild-type strain, although it took more time. These results suggest that CHS3A and CHS3B genes may have important roles in cell wall formation during protoplast regeneration, but can be compensated by other cell wall enzymes.

Phenotypes of the two temperature-sensitive actin mutants of Saccharomyces cerevisiae act1-1 and act1-2 at permissive, restrictive and semi-restrictive temperatures were studied by freeze fracture and thin section electron microscopy, and fluorescent microscopy. In contrast to secretory mutants where accumulations of either secretory vesicles, Golgi apparatus, or endoplasmic reticulum were reported, act1-1 and act1-2 mutants revealed accumulation of all the three components, even at permissive temperature. However, more distinct accumulation of secretory organelles was evident during cultivation at the sub-restrictive temperature of 30 degrees C. At the restrictive temperature of 37 degrees C, many cells died, and their empty cell walls remained. Some of the few living cells showed features of apoptosis. From the present study, actin cables are concluded to be necessary for (i) correct spatial positioning and orientation of secretary pathway to the bud and septum, and (ii) vectorial movement of vesicles of the secretory pathway along the actin cables to the bud and septum.

The morphologies, numbers, sizes and volumes of all organelles and cell components identified on ultrathin sections of aerobically grown exponential phase yeast cells of Exophiala dermatitidis in G1 phase were examined by freeze-substitution fixation and serial ultrathin sectioning. The cell wall consisted of three layers and occupied approximately 22% of the cell volume. The nucleus was approximately 1.8 microm in diameter and occupied approximately 7% of the cell volume. There was only one nucleolus in the nucleus and it occupied approximately 16% of the nuclear volume. There were 17-52 mitochondria per cell, occupying 7-12% of the cell volume. Five to ten endoplasmic reticula were present per cell; these occupied only 0.2% of the cell volume and did not form a network. There were 1-4 vacuoles per cell and they occupied 4-10% of the cell volume. Storage material was round and electron transparent and occupied 4-11% of the cell volume. The cytosol occupied 43-53% of the cell volume. The Golgi apparatus, spindle pole body, autophagosomes, multivesicular bodies, lipid bodies, microtubules and microfilaments occupied approximately 1% of the cell volume in total. About 200,000 ribosome particles, 1000 glycogen granules and several tens of microtubules (average length 0.78 microm) were present per yeast cell. The membranes of this yeast could be classified into three groups by their appearance and thickness. This is the first report, to our knowledge, that analysed all the components in the yeast cell quantitatively and in three dimensions, and provides fundamental information for understanding various aspects of cell biology.

The plasma-polymerization replica method is a unique replica technique for transmission electron microscopy. In the present study, we used this method in combination with a rapid-freeze technique to observe T4 bacteriophages and hepatitis B virus core particles. The heads of T4 bacteriophages appeared hexagonal and measured approximately 110 nm in length. Striations in their tails were also visible, indicating that the resolution of the present method is better than 4 nm. The images corresponded well with those obtained by ice-embedding and negative staining methods, with respect to both morphology and size of the phage particle. Hepatitis B virus core particles observed by the present method appeared round, approximately 30 nm in diameter, with hollow centres. Again, the morphology and size of the particles corresponded well with those obtained by ice-embedding, negative staining, and ultrathin sectioning. From these results, we conclude that the plasma-polymerization rapid-freeze replica method provides a useful technique for observation of biological specimens in a natural state and at high resolution.

In our previous transmission electron microscopic study of hepatitis B virus core antigen in transformed yeast cells, we observed core particles passing through the nuclear pores. We have now analyzed 1,421 nuclear pores in transformed yeast, and conclude that 1) translocation of core particles from the nucleus to the cytoplasm occurs through the nuclear pores; 2) translocation sites are located in the center of nuclear pores; 3) at least 95% of pores are involved in the translocation process; 4) proteins as large as 28 nm in diameter can cross the envelope; 5) translocation does not stop, but rather becomes more active during nuclear division in yeast cells.

Transformed yeast cells expressing hepatitis B virus core antigen (HBcAg) were found to accumulate abundant core particles in the same way as human hepatocytes infected with hepatitis B virus (HBV) by the present authors. We, therefore, offer a good model system for studying the dynamics of assembly of HBcAg into core particles. To investigate this problem, we have developed a transformed yeast cell in which expression of HBcAg is highly inducible by deprivation of phosphate in the culture medium. At regular intervals after induction, cells were cryo-fixed and processed for transmission electron microscopy by ultrathin sectioning. After induction, HBcAg activity rapidly increased, becoming several hundred times higher than the initial level after 25 h. The core particles first appeared in the nucleus, then in the cytoplasm, and finally in the vacuole. Core particles passing through nuclear pores from the nucleus to the cytoplasm could be seen. Core particles were either incorporated directly in the vacuole or indirectly by first forming an autophagosome. The core particles were then released into the vacuolar sap, and were digested there. Together with the previous studies, our results suggest that, in human hepatocytes, HBcAg polypeptides are synthesized in the cytoplasm, but are assembled into core particles in the nucleus. The assembled core particles are then transported from the nucleus to the cytoplasm through nuclear pores.

Methods of preparing of a new support film by plasma polymerization in a glow discharge, and its application were described. The recently developed plasma polymerization replica technique was used to prepare ultrathin films from naphthalene monomer gas on the surface of a newly cleaved sodium chloride crystal or on cover glass. The film was floated off on water or hydrofluoric acid solution to free it from the base. The plasma-polymerized naphthalene support film (PNS) prepared in this way was shown to have excellent properties by its application to negative staining of virus antigen particles and by the observation of whole ultrathin sections of animal tissue mounted on a 2 x 1 mm single slot grid. Features of the new support film are (1) its ease of preparation as a clean, very thin film of less than 10 nm, (2) its amorphous texture and high transparency to electrons, (3) its mechanical strength, resistance to heat and chemicals, and electron bombardment, and (4) its smooth and slightly hydrophilic surface. The PNS would be useful for both routine and high-resolution electron microscopy.

Flagellar (H) and somatic (O) antigens of Proteus vulgaris were differentially stained with antibodies coupled to different sizes of colloidal gold particles, and the distribution of these antigens was visualized by the plasma polymerization metal-extraction replica (PMR) method. The H antigen, labeled with 5 nm colloidal gold, was almost exclusively located on the flagella, whereas the O antigen, labeled with 10 nm colloidal gold, was almost exclusively located on the bacterial body. The marker gold particles were clearly observed as electron-dense particles on the relatively low contrast background of three-dimensional replica image of the flagellated bacteria. Thus, the PMR method may prove to be a useful tool for studying the localization of multiple substances on the cell surface, at a high resolution and in three dimensions. The diameter of the flagella measured by the replica method was about 15 nm, close to the value obtained by negative staining (16 nm). When treated with anti-flagellar (H) factor serum and protein A-gold, the diameter of flagella was significantly increased to about 35 nm. This increase in diameter was presumably caused by binding of immunoglobulins to H antigens of flagella.