Transmission and scanning electron microscopic examination of intracellular organelles in freeze‐substituted Kloeckera and Saccharomyces cerevisiae Yeast Cells

Abstract

Specimen preparation, Three-dimensional image KEY WORDS ABSTRACT The application of the conventional double-fixation method (glutaraldehyde and osmium tetroxide) to whole yeast cells is difficult because the thick cell wall of the yeast prevents the penetration of osmium tetroxide. However, this problem was solved by using the freeze-substitution fixation method. Therefore, it was possible to examine the intracellular structures of the yeast cells without digestion of the cell wall. In the present method, specimens for transmission electron microscopy and for scanning electron microscopy were prepared simultaneously. By scanning electron microscopic observation, three-dimensional information about internal structures was obtained. In the cytological analysis of the yeasts, intracellular structures were well preserved by using the freeze-substitution fixation method. On the outer leaflet of the nuclear envelope, many ribosomes were attached. The rough endoplasmic reticulum and Golgi apparatus were clearly seen in the yeast cytoplasm. The Golgi stack appeared to consist of smooth membranes, and small vesicles were present beside it. The details of other structures such as the nuclear division apparatus, actinlike filaments, and viruslike particles were also revealed. The present technique can be applied to most species of veast cells. With this new information, the previous model of a yeast cell was modified.

Ultrastructure and cytochemistry of extramural substances associated with intercellular hyphae of several rust fungi

“…aggregation leading to sporulation, this substance had a strong fibrous appearanc;e, with variations in the orientation of the fibrillar component. The layer with the perpendicularly oriented fibrils, also observed by Mendgen et al (1988), was somewhat reminiscent of the hair-like surface layer around cryoprocessed yeast cells (Baba and Osumi 1987;Kappeli et al 1984;Walther et al 1984;Tokunaga et al 1986). The fibrillar type of material predominated, but an amorphous, darker staining substance also occurred, particularly at points of cell to cell contact.…”

“…However, the structural or chemical nature structures by cryoprocessing that had not been possible with of these hyphal surfaces is not yet well understood. An outer conventional procedures (Baba and Osumi 1987;Kappeli et al layer of the intercellular hyphal wall, as detected by conven-1984;Walther et al 1984;Tokunaga et al 1986). tional processing methods for electron microscopy, has been This study was undertaken to further elucidate the occurinterpreted as a mucilaginous, possibly adhesive coating subrence and possible nature of coating substances on the interstance (Harder 1984;Littlefield and Heath 1979), but more cellular hyphae of several rust fungi, using various processing recently Chong et al (1985) supplied evidence that this layer and cytochemical techniques.…”

Mutants of Saccharomyces cerevisiae with defective vacuolar function

“…2). Figure 3 shows the ultrastructure of KL197-1A cells examined by electron microscopy by using a freeze-substitution technique (2). Cells in either the logarithmic or the stationary phase had no large central vacuole but they contained many small vesicles that were evenly distributed in the cytoplasm.…”

Efficiency and diversity of protein localization by random signal sequences.

“…Electron micrographs of yeast cells show the ER as membrane-bounded cisterna in contact with the plasma membrane and occasionally continuous with the two membranes of the nuclear envelope (17). As observed by freeze substitution electron microscopy, the ER and outer leaflet of the nuclear envelope are studded with ribosomes, as is the rough ER of mammalian cells (2). The nuclear envelope and connected ER membranes appear to be part of the same functional structure, and both have been shown to contain intracellular precursors of secreted acid phosphatase (31).…”