Electron microscopy for ultrastructural analysis and protein localization in Saccharomyces cerevisiae

Abstract

The yeast Saccharomyces cerevisiae is a key model system for studying of a multitude of cellular processes because of its amenability to genetics, molecular biology and biochemical procedures. Ultrastructural examinations of this organism, though, are traditionally difficult because of the presence of a thick cell wall and the high density of cytoplasmic proteins. A series of recent methodological and technical developments, however, has revived interest in morphological analyses of yeast (e.g. 123). Here we present a review of established and new methods, from sample preparation to imaging, for the ultrastructural analysis of S. cerevisiae. We include information for the use of different fixation methods, embedding procedures, approaches for contrast enhancement, and sample visualization techniques, with references to successful examples. The goal of this review is to guide researchers that want to investigate a particular process at the ultrastructural level in yeast by aiding in the selection of the most appropriate approach to visualize a specific structure or subcellular compartment.

Amorphous Calcium Carbonate Granules Form Within an Intracellular Compartment in Calcifying Cyanobacteria

“…Indeed, chemical fixatives and organic solvents used by conventional ultramicrotomy induced complete dissolution of intracellular ACC, which is more soluble than calcite ( Li et al, 2016 ). Several methods of biological sample preparation alternative to conventional ultramicrotomy have been previously developed (e.g., Edelmann, 2002 ; Cavalier et al, 2009 ; Frankl et al, 2015 ). For example, freeze-substitution has been shown to preserve cell ultrastructures very well, including membranes, the cytoplasm and DNA (e.g., Giddings, 2003 ; Bleck et al, 2010 ).…”

2D and 3D immunogold localization on (epoxy) ultrathin sections with and without osmium tetroxide

“…Furthermore it was shown, that higher concentrations of OsO 4 could have a cell component destructive, proteolytic, and actin filament degrading effect (Baschong et al, ; Behrman, ; Locke, ; Maupin & Pollard, ; Maupin‐Szamier & Pollar, ). Therefore, and if it cannot be avoided as supposed by Hopwood () and others (Frankl et al, ), a reduction of the OsO 4 concentration makes sense. However, the application of OsO 4 ‐concentrations in the range of 0.2 or 0.3% instead of 2% in freeze substitution significantly reduces the negative effect of the chemical but still enables very good structural details (Liu et al, ; Maupin & Pollard, ; Mix et al, ; Peschke et al, ).…”

“…The experiments shown in this study, but also in previous studies from our laboratory demonstrated that Epon sections can be superior for many immunolabeling studies, at least when studying archaeal cells and microalgal cells (Heimerl et al, ; Küper et al, ; Liu et al, ; Mayer et al, ; Meyer, Heimerl, Wirth, Klingl, & Rachel, ; Mix et al, ; Peschke et al, ; Schreiber et al, ). Slight changes in the chemical nature of nowadays resins compared to the 1970s and 1980s (e.g., Embed 812 from Electron Microscopy Sciences as replacement product for EPON 812; Luft, ) make epoxy resins a lot more useful than generally thought (Frankl, Mari, & Reggiori, ; Stirling, ). However, it needs to be noted that we were not able to detect gold particles on Epon sections with three out of six tested primary antibodies (Table ).…”

Correlative light and electron microscopy methods for the study of virus–cell interactions

“…Active replication of SFV genome is indicated by the coexpression of red and green fluorescent proteins (1)(2)(3). The cell of interest is first identified and imaged by confocal microscope in both fluorescent and transmitted light channels to record the position on the grid and then further analyzed by TEM (5)(6)(7)(8). Clusters of spherules were identified at the bottom of cells (5-7) and on the cell membrane (8).…”

“…The cell of interest is first identified and imaged by confocal microscope in both fluorescent and transmitted light channels to record the position on the grid and then further analyzed by TEM (5)(6)(7)(8). Clusters of spherules were identified at the bottom of cells (5-7) and on the cell membrane (8). Images in (A) are unpublished data from Cortese M. and Bartenschlager R.; figures in (B) and (C) are modified from refs.…”