The ultrastructure of yeast: Cell wall structure and formation

To identify new genes involved in the control of cell morphogenesis in the fission yeast Schizosaccharomyces pombe we have visually screened for temperature-sensitive mutants that show defects in cell morphology. We have isolated and characterized 64 mutants defining 19 independent genes, 10 of which have not been previously described. One class of mutants, defining 12 orb genes, become round and show a complete loss of cell polarity. A second class of mutants exhibits branched or bent morphologies. These mutants show defects in either selection of the growth site, defining two tea genes, or in the maintenance of growth direction, defining five ban genes. Immunofluorescence analysis of these morphological mutants shows defects in the organization of the microtubule and actin cytoskeleton. These defects include shortened, bundled, and asymmetrically localized microtubules and enlarged and mislocalized actin patches. Analysis of the mutant phenotypes has allowed us to order the genes into four groups according to their function during the cell cycle: genes required for the maintenance of cell polarity throughout the cell cycle; genes necessary only for the reestablishment of cell polarity after mitosis and not for maintaining cell polarity once it is established; genes essential for the transition from monopolar to bipolar growth and genes that severe as 'polarity markers'.

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Fission yeast cell morphogenesis: Identification of new genes and analysis of their role during the cell cycle

The fission yeast, Schizosaccharomyces pombe, has been used as a model eukaryote to study processes such as the cell cycle and cell morphology In this single-celled organism, growing in a straight line and maintaining the nucleus in the centre of the cell depend on intracellular positional information Microtubules and microtubular transport are important for generating positional information within the fission yeast cell, and these molecular mechanisms are also probably relevant for generating positional information in other eukaryotic cells

A journey into space.

Cryptococcal meningitis is a fungal infection, caused by Cryptococcus neoformans, which is prevalent in immunocompromised patient populations. Treatment failures of this disease are emerging in the clinic, usually associated with long-term treatment with existing antifungal agents. The fungal cell wall is an attractive target for drug therapy because the syntheses of cell wall glucan and chitin are processes that are absent in mammalian cells. Echinocandins comprise a class of lipopeptide compounds known to inhibit 1,3-beta-glucan synthesis, and at least two compounds belonging to this class are currently in clinical trials as therapy for life-threatening fungal infections. Studies of Saccharomyces cerevisiae and Candida albicans mutants identify the membrane-spanning subunit of glucan synthase, encoded by the FKS genes, as the molecular target of echinocandins. In vitro, the echinocandins show potent antifungal activity against Candida and Aspergillus species but are much less potent against C. neoformans. In order to examine why C. neoformans cells are less susceptible to echinocandin treatment, we have cloned a homolog of S. cerevisiae FKS1 from C. neoformans. We have developed a generalized method to evaluate the essentiality of genes in Cryptococcus and applied it to the FKS1 gene. The method relies on homologous integrative transformation with a plasmid that can integrate in two orientations, only one of which will disrupt the target gene function. The results of this analysis suggest that the C. neoformans FKS1 gene is essential for viability. The C. neoformans FKS1 sequence is closely related to the FKS1 sequences from other fungal species and appears to be single copy in C. neoformans. Furthermore, amino acid residues known to be critical for echinocandin susceptibility in Saccharomyces are conserved in the C. neoformans FKS1 sequence.

A glucan synthase FKS1 homolog in cryptococcus neoformans is single copy and encodes an essential function.

The challenges of sample preparation can limit a researcher's selection of transmission electron microcopy (TEM) for analysis of yeast. However, with the exception of thin sectioning, preparation of well-fixed and infiltrated samples of yeast cells is achievable by any reasonably equipped laboratory. This review presents a general overview of TEM sample preparation methods and detailed protocols for chemical fixation of yeast for ultrastructural analysis and immunolabeling. For ultrastructural analysis, the most commonly used chemical fixation involves treatment with glutaraldehyde followed by either potassium permanganate or osmium. Prior to osmium postfixation, the cell wall must be enzymatically digested to allow optimal fixation and embedding. Freeze substitution methods continue to provide the highest quality of fixation, but equipment needed for these protocols is not generally available to many labs. The low viscosity of Spurr's resin makes it the resin of choice for ultrastructure studies. Immunoelectron microscopy has enjoyed great success in analysis of yeast molecular organization. For immunoelectron microscopy, glutaraldehyde/formaldehyde-fixed cells are embedded in LR White resin. The thin sections are then treated in much the same way as an immunoblot: following blocking, they are incubated in primary antiserum, washed, and then incubated in gold-labeled secondary antiserum.

Transmission electron microscopy of yeast.

To clarify the involvement of actin in the formation of the yeast cell wall, reverting protoplasts of Schizosaccharomyces pombe were used as a simple model system. Actin of reverting protoplasts was labeled with rhodamine-conjugated phalloidin and observed by conventional fluorescence microscopy and laser scanning confocal microscopy. A close spatial as well as temporal relationship between actin and cell wall formation was observed in protoplast reversion. That is, the site of actin 'dots' in the reverting protoplasts coincided with the site of new wall formation and the timing of rearrangement of actin coincided with the initiation of cell wall formation and with the timing of cell wall expansion. Treatment of reverting protoplasts with cytochalasin D (CD) further clarified the close relationship between actin and cell wall organization. The effect of CD was dose dependent. A high dose of CD caused the absence of actin as well as the complete inhibition of cell wall formation. A low dose of CD caused weakly stained unlocalized actin, which induced grossly aberrant cell wall deposition as well as substantial changes in the morphology of the reverting protoplasts. These results demonstrated that actin is associated with initiation of cell wall formation, the proper deposition of cell wall materials, and maintaining the normal morphology of reverting protoplasts. Scanning electron microscopy revealed the presence of a fibrillar net structure on the surface of non-treated control reverting protoplasts. However, the absence of a fibrillar network on the surface of reverting protoplasts was observed in the presence of a high concentration of CD. Lack of localization of microfibrils as well as poor development of the fibrillar network were also observed in the presence of a low concentration of CD. Recovery experiments confirmed the close relationship between actin and cell wall formation.

Actin is associated with the formation of the cell wall in reverting protoplasts of the fission yeast Schizosaccharomyces pombe.

The ultrastructure of regenerating cell wall in Schizosaccharomyces pombe protoplasts was studied with a high resolution, low voltage scanning electron microscope (LVSEM). In contrast to the transmission electron microscopy, the LVSEM images give three-dimensional information on the cell wall regeneration in yeast protoplasts. We found that, after only a few minutes of incubation, the protoplasts began to show protuberances in a unipolar manner, and a fibrilar network was formed asymmetrically which covered the whole surface of the protoplasts after 5 hr. The network consisted of microfibrils about 8 to 10 nm wide, forming flat and wavy bundles of various widths and lengths, up to about 200 nm wide and 1 micron long, mainly made of yeast glucan. Free ends of microfibrils were seldom found. Interfibrillar spaces were progressively filled with granular particles and finally the complete cell wall was formed after 12 hr. The fibrillar network was destroyed by the digestion with beta (1----3)-glucanase. When protoplasts were regenerating in the presence of aculeacin A, the fibrillar networks were not formed, resulting in incomplete cell wall formation. These observations suggest that beta-glucan is the main component of the microfibrils and that it plays an important role in the formation of the cell wall in S. pombe.

Cell Wall Formation in Regenerating Protoplasts of Schizosaccharomyces pombe: Study by High Resolution, Low Voltage Scanning Electron Microscopy

High Resolution, Low Voltage Scanning Electron Microscopy of Uncoated Yeast Cells Fixed by the Freeze-Substitution Method

Trichophyton mentagrophytesの形態および微細構造に及ぼすNND-318の影響を走査型電子顕微鏡および透過型電子顕微鏡を用いて観察した.段階的濃度の薬剤を含む培地にT.mentagrophytes分生子を接種し,27℃で60時間スライド培養した.薬剤無添加では菌糸はほぼ直線状に伸長していたが,NND-318 0.08～0.4ng/ml添加では菌糸の屈曲,過剰分岐,菌糸幅の不均一化および皺襞形成等の形態変化が認められた.菌糸細胞内では,細胞壁の肥厚および壁内の顆粒状構造物出現,ならびに細胞質内の脂肪滴の増加が薬剤濃度の上昇に伴い認められた.0.8～1.6ng/mlでは菌糸の皺襞は更に明瞭となり,伸長は著しく阻害されていた.4.0ng/ml以上の濃度では菌糸形成は認められず分生子の形態を留めていた.比較対照のために用いたbifonazoleもまたより高濃度域ではあるがNND-318と同質の形態異常を引き起こすことが観察された.以上の結果からNND-318は従来のイミダゾール剤よりも著しく低濃度でT.mentagrophytes分生子の発芽ならびに菌糸の伸長発育を阻害すること,および発育を阻止された菌糸細胞においては細胞質内への脂肪滴の蓄積,細胞壁の肥厚,細胞壁内の顆粒状構造物形成促進などの超微形態学的変化が引き起こされることが示された.

Akiko Taki

Papers

Actin is associated with the formation of the cell wall in reverting protoplasts of the fission yeast Schizosaccharomyces pombe.

Cell Wall Formation in Regenerating Protoplasts of Schizosaccharomyces pombe: Study by High Resolution, Low Voltage Scanning Electron Microscopy

High Resolution, Low Voltage Scanning Electron Microscopy of Uncoated Yeast Cells Fixed by the Freeze-Substitution Method

Effect of NND-318 on the Ultrastructure of Trichophyton mentagrophytes as Observed by Scanning and Transmission Electron Microscopy

Structural Effects of Sulconazole Nitrate on the Hyphal Growth of Trichophyton rubrum