| Research Abstract |
1) The UDP-galactose transporter deletion mutant which is deficient in α-galactomannan formation showed a shorter-brush structure of the outermost layer compared to the wild type cells. In contrast, cpsl, the β-1,3-glucan synthase mutant, and mokl, the α-glucan synthase mutant, were round in shape. Both mutants had a thick and loose cell wall. The glucan network was exposed on the surface only in mokl. These suggested that the α-galactomannan was responsible for the formation of the surface structure, and thatα- and β-1,3-glucans were necessary as a frame in the cell wall construction to maintain the normal cylindrical cell shape.
2) The cpsl cell seemed abnormal from the first stage of the primary septum formation, but the mokl formed a thick secondary septum. These suggested that β-1,3-glucan was important for the primary septum formation and that α-1,3-glucan was required in the secondary septum formation.
3) By fluorescent microscopy, the linear-β-1,3-glucan and low molecule α-1,3-gl
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ucan appeared in the area of the newly formed cell wall from the beginning of protoplast regeneration. The linear-β-1,3-glucan was located on the glucan network, but the α-1,3-glucan was seen near the cell membrane.
4) By IEM, α-1,3-glucan localized on the adjacent side of the cell membrane in the septa until the cell divided; this differed from the linear β-1,3-glucan which was located on the primary septum. Both these glucans appeared from the beginning of the glucan formation, but β-1,6-glucan did not appear, indicating that each glucan had a specific role in the glucan network formation.
5) Bgslp, one of the β- 1,3-glucan synthases, located on the cell membrane in the septum region until the completion of septum true of Moklp, the former was only seen at the tip of the primary septum. Bgslp accumulated in the electron dense area at the medial region before completion of the nuclear division. These findings mean that not α-1,3-glucan but β-1,3-glucan played a vital role in the primary septum formation and that β-1,3-glucan also acted in the starting of the primary formation.
6) By SEM, purified α-1,3-glucan was a fiber about 10 to 20-nm in diameter, and had a mesh-like shape. The cell treated with an alkaline reagent in which only three glucans remained, retained a cylindrical shape and its surface was covered with the network structure. However, after resolution of β-1,3-glucan and β-1,6-glucan, the cell became flat and could not keep its cylindrical shape. These findings indicated that α-1,3-glucan maintained the specific shape of the fission yeast combining with β-glucans to form the network.
7) The deletion mutant of β-1,6-glucan synthesis related gene, Δ rotl, was a short cylinder and was round. This mutant had an uneven cell surface on which α-galactomannan particles could not cover the network structure. The cell wall materials were abnormally accumulated inside the cell. This suggests a possibility that the β-1,6-glucan is concerned with accumulation of α-galactomannan in the glucan network.
8) The septum formation in Δ rotl was abnormal, although the primary septum was formed normally. The two septa were formed into a cell, sometimes forming an X-shape septum. This indicates that the role of β-1,6-glucan was different from that of α-1,3-glucan in the secondary-septum formation.
9) To visualize by EM the polarity of F-actin in the cell by decorating it with a myosin head to form an arrowhead structure, the cell was enzymically digested and permeabilized with detergent so that myosin S1 could penetrate the cytoplasm. We tried this in fission yeast which enabled us to analyze the transportation of cell wall materials. Less
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