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In fission yeast, Schizosaccharomyces pombe, the carbohydrate components of the cell wall consist of galactomannan, unlike in Saccharomyces cerevisiae. We previously found that the disruption of gms1⁺, a gene encoding the UDP-galactose transporter required for the synthesis of galactomannan, led to the complete defect of cell surface galactosylation in Sz. pombe. The Δgms1 strain is therefore useful for the analysis of physiological properties of galactose residues in Sz. pombe. The deletion strain of gms1⁺ was viable; however, itshowed an aberrant cell morphology and increased sensitivities to digestion with β-glucanase and to various drugs, such as hygromycin B, sodium orthovanadate and Calcofluor white. A reduction of galactomannan layers of the cell wall in the Δgms1 strain was observed by scanning and transmission electron microscopic analyses. The addition of osmotic stabilizer suppressed the morphologic defect of the Δgms1 cells, while other phenotypes were weakly suppressed. The Δgms1 (h⁹⁰) strain was incapable of sexual conjugation during nutritional starvation. These results suggest that the cell surface galactosylation is required not only for non-sexual flocculation but also for sexual conjugation in Sz. pombe. Copyright © 2001 John Wiley & Sons, Ltd.

In fission yeast, Schizosaccharomyces pombe, the carbohydrate components of the cell wall consist of galactomannan, unlike in Saccharomyces cerevisiae. We previously found that the disruption of gms1+, a gene encoding the UDP‐galactose transporter required for the synthesis of galactomannan, led to the complete defect of cell surface galactosylation in Sz. pombe. The Δgms1strain is therefore useful for the analysis of physiological properties of galactose residues in Sz. pombe. The deletion strain of gms1+was viable; however, itshowed an aberrant cell morphology and increased sensitivities to digestion with β‐glucanase and to various drugs, such as hygromycin B, sodium orthovanadate and Calcofluor white. A reduction of galactomannan layers of the cell wall in the Δgms1strain was observed by scanning and transmission electron microscopic analyses. The addition of osmotic stabilizer suppressed the morphologic defect of the Δgms1cells, while other phenotypes were weakly suppressed. The Δgms1(h90) strain was incapable of sexual conjugation during nutritional starvation. These results suggest that the cell surface galactosylation is required not only for non‐sexual flocculation but also for sexual conjugation in Sz. pombe. Copyright © 2001 John Wiley & Sons, Ltd.

The chemical composition of the cell wall of Sz. pombe is known as β-1,3-glucan, β-1,6-glucan, α-1,3-glucan and α-galactomannan; however, the three-dimensional interactions of those macromolecules have not yet been clarified. Transmission electron microscopy reveals a three-layered structure: the outer layer is electron-dense, the adjacent layer is less dense, and the third layer bordering the cell membrane is dense. In intact cells of Sz. pombe, the high-resolution scanning electron microscope reveals a surface completely filled with α-galactomannan particles. To better understand the organization of the cell wall and to complement our previous studies, we set out to locate the three different types of β-glucan by immuno-electron microscopy. Our results suggest that the less dense layer of the cell wall contains mainly β-1,6-branched β-1,3-glucan. Occasionally a line of gold particles can be seen, labelling fine filaments radiating from the cell membrane to the α-galactomannan layer, suggesting that some of the radial filaments contain β-1,6-branched β-1,3-glucan. β-1,6-glucan is preferentially located underneath the α-galactomannan layer. Linear β-1,3-glucan is exclusively located in the primary septum of dividing cells. β-1,6-glucan only labels the secondary septum and does not co-localize with linear β-1,3-glucan, while β-1,6-branched β-1,3-glucan is present in both septa. Linear β-1,3-glucan is present from early stages of septum formation and persists until the septum is completely formed; then just before cell division the label disappears. From these results we suggest that linear β-1,3-glucan is involved in septum formation and perhaps the separation of the two daughter cells. In addition, we frequently found β-1,6-glucan label on the Golgi apparatus, on small vesicles and underneath the cell membrane. These results give fresh evidence for the hypothesis that β-1,6-glucan is synthesized in the endoplasmic reticulum–Golgi system and exported to the cell membrane. Copyright © 2001 John Wiley & Sons, Ltd.

The chemical composition of the cell wall of Sz. pombeis known as β‐1,3‐glucan, β‐1,6‐glucan, α‐1,3‐glucan and α‐galactomannan; however, the three‐dimensional interactions of those macromolecules have not yet been clarified. Transmission electron microscopy reveals a three‐layered structure: the outer layer is electron‐dense, the adjacent layer is less dense, and the third layer bordering the cell membrane is dense. In intact cells of Sz. pombe, the high‐resolution scanning electron microscope reveals a surface completely filled with α‐galactomannan particles. To better understand the organization of the cell wall and to complement our previous studies, we set out to locate the three different types of β‐glucan by immuno‐electron microscopy. Our results suggest that the less dense layer of the cell wall contains mainly β‐1,6‐branched β‐1,3‐glucan. Occasionally a line of gold particles can be seen, labelling fine filaments radiating from the cell membrane to the α‐galactomannan layer, suggesting that some of the radial filaments contain β‐1,6‐branched β‐1,3‐glucan. β‐1,6‐glucan is preferentially located underneath the α‐galactomannan layer. Linear β‐1,3‐glucan is exclusively located in the primary septum of dividing cells. β‐1,6‐glucan only labels the secondary septum and does not co‐localize with linear β‐1,3‐glucan, while β‐1,6‐branched β‐1,3‐glucan is present in both septa. Linear β‐1,3‐glucan is present from early stages of septum formation and persists until the septum is completely formed; then just before cell division the label disappears. From these results we suggest that linear β‐1,3‐glucan is involved in septum formation and perhaps the separation of the two daughter cells. In addition, we frequently found β‐1,6‐glucan label on the Golgi apparatus, on small vesicles and underneath the cell membrane. These results give fresh evidence for the hypothesis that β‐1,6‐glucan is synthesized in the endoplasmic reticulum–Golgi system and exported to the cell membrane. Copyright © 2001 John Wiley & Sons, Ltd.

A Schizosaccharomyces pombe cps8mutant, of which the gene encodes a mutant actin with an amino acid substitution of Asp for Gly273[J. Ishiguro and W. Kobayashi (1996) FEBS Lett. 392, 237–241], was used to determine the role of the actin cytoskeleton in cell wall formation. In the cps8mutant cells, atomic force microscopic and scanning electron microscopic images showed abnormal depolarized and branched morphology. Fibrous material covered a part of the surface of growing cps8cells. Transmission electron microscopic images showed variable thickness of the cell wall due to multilayering of cell wall materials, and aberrant multisepta due to diagonal growth of the primary septum, whereas the normal primary septum grows at a right angle from the cortex. This abnormal septum formation may induce abnormality of the cell with multinuclei and/or multisepta, caused by non‐separation of daughter cells. These results indicate that actin plays an important role in cell wall and septum formation.

Yeast cells have a thick cell wall composed of an inner network of glucans and an outer layer of mannoproteins, which is difficult to penetrate with osmium tetroxide. We previously developed the sandwich technique to overcome this problem. Although the freeze-etching method allows the fracturing of cryofixed yeast cells, it has been difficult to fracture cryofixed yeast cells for examination by cryo-scanning electron microscopy (SEM). The development of an alternative method of cryofixation, namely, high-pressure freezing, began in the 1960s and is now available for the electron microscopic analysis of yeast. We show here that when high-pressure freezing is combined with ultra-low temperature and low-voltage SEM using the new cryo-system, the Gatan Alto 2500 Cryo Transfer System, fractured and coated yeast samples could be quickly prepared. These samples yielded a fine fracture plane and revealed the ultrastructure of both external and internal cell components. We used this method to analyze the process of septum formation, one of the final and most important events of mitosis, and cell separation. The images we obtained provide a three-dimensional view of these processes for the first time. We also showed that high-pressure freezing in combination with immunoelectron microscopy made it possible to preserve the antigenicity, in situ localization, and behavior of the cell wall component α-1,3-glucan and its synthase during septum formation in Schizosaccharomyces pombe.

We aimed at investigating the continuity of the Golgi apparatus into the endoplasmic reticulum (ER) irs cultured tobacco protoplasts by zinc iodide-osmium (ZTO) staining. The density of the membranes of both Golgi cisternae and the ER was, at first analysed by measuring their peak contrast on software:NIH Image 1.60. Membranes of the Golgi cisternae were differen tially densely stained when the protoplasts were immersed in the ZTO mixture for 16 h. These Golgi membranes were more dearly identified than those stained with uranyl acetate alone. After treatment with 10 μg ml−1 brefeldin A (BFA) for 3 h, the distorted Golgi cistemae became wider and longer than those in control protoplasts. The expanded cisternae were loosely organized and their tips appeared to extend into the ER. In ZIO stained sections, the density (the mean value of peak contrast) of the membranes of the Golgi cisternae elongated to the ER were the same as those of the ER after treatment of the drug for 4 h. These results indicate that the extended cistemae of the Golgi apparatus continued into the ER after 3 h treatment of BFA.