In situ localization of beta-glucans in the cell wall of Schizosaccharomyces pombe

The chemical composition of the cell wall of Sz. pombe is known as beta-1,3-glucan, beta-1,6-glucan, alpha-1,3-glucan and alpha-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 alpha-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 beta-glucan by immuno-electron microscopy. Our results suggest that the less dense layer of the cell wall contains mainly beta-1,6-branched beta-1,3-glucan. Occasionally a line of gold particles can be seen, labelling fine filaments radiating from the cell membrane to the alpha-galactomannan layer, suggesting that some of the radial filaments contain beta-1,6-branched beta-1,3-glucan. beta-1,6-glucan is preferentially located underneath the alpha-galactomannan layer. Linear beta-1,3-glucan is exclusively located in the primary septum of dividing cells. beta-1,6-glucan only labels the secondary septum and does not co-localize with linear beta-1,3-glucan, while beta-1,6-branched beta-1,3-glucan is present in both septa. Linear beta-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 beta-1,3-glucan is involved in septum formation and perhaps the separation of the two daughter cells. In addition, we frequently found beta-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 beta-1,6-glucan is synthesized in the endoplasmic reticulum-Golgi system and exported to the cell membrane.