Differential cell wall remodeling of two chitin synthase deletants Δchs3A and Δchs3B in the pathogenic yeast Candida glabrata.

It is known that cell wall remodeling and the salvaging pathway act to compensate for an impaired or a damaged cell wall. Lately, it has been indicated that this mechanism is partly required for resistance to the glucan synthesis inhibitor echinocandin. While cell wall remodeling has been described in mutants of glucan or mannan synthesis, it has not yet been reported in a chitin synthesis mutant. Here, we describe a novel cell wall remodeling and salvaging pathway in chitin synthesis mutants, Δchs3A and Δchs3B, of the pathogenic yeast Candida glabrata. Electron microscopic analysis revealed a thickened mannoprotein layer in Δchs3A cells and a thickened chitin-glucan layer of Δchs3B cells, and it indicated the hypothesis that mannan synthase and chitin-glucan synthase indemnify Δchs3A and Δchs3B cells, respectively. The double-mutant CHS3A and MNN10, encoding α-1,6-mannosyltransferase, showed synergistic stress sensitization, and the Δchs3B strain showed supersensitivity to echinocandins. Hence, these findings support the above hypothesis of remodeling. Furthermore, unlike Δchs3A cells, Δchs3B cells showed supersensitivity to calcineurin inhibitor FK506 and Tor1p kinase inhibitor rapamycin, indicating that the Δchs3B strain uses the calcineurin pathway and a Tor1p kinase for cell wall remodeling.

Improved preservation of fine structure of deep-sea microorganisms by freeze-substitution after glutaraldehyde fixation.

A method was proposed for improving preservation of ultrastructures of deep-sea microorganisms by using rapid-freeze freeze-substitution after glutaraldehyde fixation. This method produced clear high-resolution images of cells appearing in their natural state, close to the quality of images obtained by rapidly freezing freeze-substituted specimens of living cells. The method may be useful for observing any microorganism when rapid freezing of living samples is difficult and only glutaraldehyde fixation can be carried out.

Scanning and negative-staining electron microscopy of protoplast regeneration of a wild-type and two chitin synthase mutants in the pathogenic yeast Candida glabrata.

Protoplast regeneration of a wild-type and two mutant strains of Candida glabrata defective in CHS3 homologues encoding class IV chitin synthase in Saccharomyces cerevisiae was examined by scanning and negative-staining electron microscopy. In the wild-type strain, small particles and short filaments appeared on the protoplast surface at 10 min, filamentous materials covered the entire surface of the protoplast at 1 h, granular materials started filling interspaces of filamentous materials at 2 h and regeneration was completed at 6 h. The filamentous materials consisted of microfibrils of various widths ranging from ≤5 to 40 nm, and composed of ß-glucan. Protoplasts of the two chitin synthase mutant strains of Δchs3A and Δchs3B completed regeneration essentially by the same process as wild-type strain, although it took more time. These results suggest that CHS3A and CHS3B genes may have important roles in cell wall formation during protoplast regeneration, but can be compensated by other cell wall enzymes.

Structome of Saccharomyces cerevisiae determined by freeze-substitution and serial ultrathin-sectioning electron microscopy.

The cell structure has been studied using light and electron microscopies for centuries, and it is assumed that the whole structure is clarified by now. Little quantitative and three-dimensional analysis of cell structure, however, has been undertaken. We have coined a new word, 'structome', by combining 'structure' and '-ome', and defined it as the 'quantitative and three-dimensional structural information of a whole cell at the electron microscopic level'. In the present study, we performed structome analysis of Saccharomyces cerevisiae, one of the most widely researched biological materials, by using freeze-substitution and serial ultrathin-sectioning electron microscopy. Our analysis revealed that there were one to three mitochondria, ~220 000 ribosomes in a cell, and 13-28 endoplasmic reticula/Golgi apparatus which do not form networks in the cytoplasm in the G1 phase. Nucleus occupied ~10.5% of the cell volume; cell wall occupied ~17%; vacuole occupied ~5.8%; cytoplasm occupied ~64%; and mitochondria occupied only ~1.7% in the G1 phase. Structome analysis of cells would form a base for the post-genome research.

Smart specimen preparation for freeze substitution and serial ultrathin sectioning of yeast cells.

A smart and efficient method for freeze substitution and serial sectioning of yeast cells is described. Yeast cells were placed in a single layer between two copper disks, rapidly frozen, freeze substituted and embedded in an epoxy resin. The cell layer was re-embedded by the same resin, the surface trimmed leaving 1 mum above the cell layer, and serially sectioned. The sections were collected on the two-slit grids and placed on a Formvar film mounted to cover the holes of an aluminum supporting rack. The grids were removed from the rack, stained together using a silicon tube and observed in a transmission electron microscope. The images of yeast cells observed were clear and natural, and would be useful for a detailed 3D structural analysis such as structome.