The ultrastructure of yeast: Cell wall structure and formation

Yeasts are unicellular eukaryotes, and are used widely as a model system in basic and applied fields of life science, medicine, and biotechnology. The ultrastructure of yeast cells was first studied in 1957 and the techniques used have advanced greatly in the 40 years since then; an overview of thes e methods is first presented in this review. The ultrastructure of budding and dimorphic yeast cells observed with a scanning electron microscope (SEM ) and a transmission electron microscope (TEM) after thin sectioning and fr eeze-etching are then described, followed by discussion of the regeneration of the cell wall of Candida albicans protoplasts detected by cryosectionin g. C. albicans protoplasts are regenerated to synthesize microfibrils on th eir surface. They are aggregated into thicker bundles which an intermeshed, forming a wide-meshed network of long fibrils. These microfibrillar struct ures are chains of beta-1,3-glucan which are broken down after treatment wi th beta-1,3-glucanase. Morphologically identical microfibrils are synthesiz ed in vitro by a cell-free system in which the active cell membrane fractio n as a source of beta-1,3-glucan synthetase and UDP glucose as the sole sub strate are used. The diameter of an elemental fibril of beta-glucan is esti mated to be: 2.8 nm from the pattern of autocorrelation of the image obtain ed by computer processing. In contrast, in the presence of aculeacin A the formation of normal fibrillar nets or bundles is significantly inhibited, r esulting in the occurrence of short fibrils. These electron microscopic dat a suggest that aculeacin A inhibits not only the synthesis of beta-1,3-gluc an but the aggregation of microfibrils of this polysaccharide, allowing for mation of the crystalline structure. On the basis of the cumulative data ob tained from the electron microscopic studies, we are led to the assumption that de novo synthesized beta-glucan chains might initially form fine parti cles which are then transformed into thin fibrils with single to multiple s trands which appear to be oriented parallel to each other so that they deve lop into fibrillar structures. This process of assembly of beta-glucan mole cules leads to the development of a fibrous network within the regenerating Candida cell wall. Third, the mechanism of cell wall formation is shown by low-voltage (LV) SEM and TEM, using various techniques and computer graphi cs, of the regeneration system of Schizosaccharomyces pombe protoplasts: af ter 10 min of regeneration, the protoplasts begin to grow fibrillar substan ces of a beta-glucan nature, and a fibrillar network covers the surface of all protoplasts. The network is originally formed as fine particles on the protoplast surface and these are subsequently lengthened to microfibrils 2 nm thick. The microfibrils twist around each other and develop into 8 nm th ick fibrils forming Rat bundles 16 nm thick. Interfibrillar spaces are grad ually filled with amorphous particles of an alpha-galactomannan nature and, finally, the complete cell wall is formed after 12 h. Treatment of reverting protoplasts with RuO4 provided clear TEM images of g lucan fibrils with high electron density. The relationship between cell wal l regeneration and intracellular organelles was examined by using serial th in sections stained with PATAg and computer-aided three-dimensional reconst ruction. The secretory vesicles in a protoplast had increased markedly by 1 .4, 3.3, and 5.8 times at 1.5, 3.0. and 5 h, respectively. Three-dimensiona l analysis indicates that Golgi apparatuses are located close together in t he nucleus of the protoplast and are dispersed into the cytoplasm during th e progress of cell wall formation. (C) 1998 Elsevier Science Ltd. All right s reserved.