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'.

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.

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.

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 β-glucan.

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.