Structome Analysis Of The Yeast Saccharomyces Determined By Freeze-substitution And Serial Ultrathin Sectioning Electron Microscopy

The cell structure has been studied by light and electron microscopy for centuries, and it is assumed that the whole structure is clarified by now. However, little quantitative and three-dimensional analysis of cell structure has been undertaken, and the number of ribosomes or the number and cellular distribution of endoplasmic reticula in a yeast cell are not known. Dr. Masashi Yamaguchi 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.’

Saccharomyces cerevisiae was used for the first genome analysis in eukaryotes and is one of the most common organisms studied in cell biology worldwide. Since genetic, biochemical, and morphological information on this material has accumulated, quantitative and three-dimensional structural information on the whole cell at the electron microscopic level, i.e., the structome, would provide useful additional information.

S. cerevisiae cells were cultured in 20 ml of YPD liquid medium (1% yeast extract, 2% polypeptone, and 2% glucose) at 30°C, and exponentially growing cells were used for structome analysis. Cells were collected by centrifugation, sandwiched between two copper disks and snap frozen by plunging into melting propane cooled with liquid nitrogen. They were freeze-substituted in acetone containing 2% osmium tetroxide at -80°C, and embedded in epoxy resin. Serial ultrathin sections were cut to a thickness of 90 nm with a diamond knife, picked up on slit grids, and stained with uranyl acetate and lead citrate. The micrographs were taken at a magnification of 10,000x in a JEM1200EX electron microscope (JEOL, Tokyo, Japan). Thirty-two complete serial sections of S. cerevisiae cells were photographed (Fig. 1) and six cells were chosen and used for structome analysis. Three cells were single unbudded cells and the other three were daughter cells that finished cytokinesis but were still attached to mother cells.

Fig. 1. Serially sectioned yeast cells from total of 45 sections. The number indicates the section number. Note that the images were clear and natural. N, nucleus; V, vacuole. (Reprinted from Yamaguchi et al., J Electron Microsc 58, 261-266, 2009)

Fig. 2. High magnification images of various cell components in S. cerevisiae.

a The cell wall (CW) and plasma membrane (PM). O, outer cell wall layer; I, inner cell wall layer. ER, endoplasmic reticulum. b Invagination (Inv). c Mitochondrion (M) with outer membrane (OM). d Autophagosome (A). e Multivesicular body (MVB). f Peroxisome (P). g Endoplasmic reticulum/Golgi apparatus (ER/Golgi). h Virus–like particles (VLP). i Nucleus (N), Nuclear envelope (NE), Nuclear pore (NP), and Nucleolus (Nu). j Ribosomes (R). k Filasome (F) l Small vesicles (Ves). m Spindle pole body (SPB), Microtubule (Mt). n Microfilaments (Mf). o Vacuole (V) with vacuolar membrane (VM). Note clear and natural morphology of each cell component. Bar = 100 nm. (Reprinted from Yamaguchi et al., J Electron Microsc 60, 337-351, 2011)

Although preparing serial ultrathin sections is considered to be very difficult, it is rather easy if the proper method is used. Drs. Yamaguchi and Chibana show a step-by-step procedure for safely obtaining serial ultrathin sections of microorganisms. This new method enables obtaining serial ultrathin sections without any difficulty. The method makes it possible to analyze cell structures of microorganisms at high resolution in 3D, which cannot be achieved by using the automatic tape-collecting ultramicrotome method and serial block face or focused ion beam scanning electron microscopy.

S. cerevisiae cells were nearly spherical ellipsoids, 3.24 µm in the minor axis and 3.85 µm in the major axis.The volume was 15.2 µm3. The cell wall was 120 nm thick, consisted of two layers (Fig. 2a), and occupied 18 % of the cell volume (Fig. 3). The nucleus was enclosed by a double layered nuclear envelope (Fig. 2i), 1.63 µm in diameter, and 1.5 µm3 in volume occupying 10 % of the cell volume. The mitochondria appeared oval or elongated in ultrathin sections (Fig. 2c) and were found to be string-shaped and sometimes branched in 3D reconstruction images (Fig. 4). The mitochondria had a total volume of 0.23 µm3 and occupied 2 % of the cell volume. There was one giant mitochondrion in most cells. There were 20 ER/Golgi in a cell with a total volume of 0.09 µm3 occupying only 0.6 % of the cell volume. The ER/Golgi in Saccharomyces exists mostly like islet and does not form network. The total vacuolar volume was 0.68 µm3 and occupied 4 % of the cell volume. The total number of ribosome particles in S. cerevisiae cells were 200,000. The number per unit volume of cytosol was 20,000/µm3. Cytosol occupied 66 % of the cell volume (Fig. 3).

Fig. 3. Proportions of the cytosol (C), cell wall (CW), nucleus (N), mitochondria (M), vacuoles (V), and other cell components (O) in 6 cells of S. cerevisiae. Values are expressed as percent to the cell volume. ‘Others’ include endoplasmic reticulum/Golgi apparatus, autophagosomes, and multivesicular bodies. The volume of cytosol includes the volume of ribosome particles, small vesicles, and virus-like particles. (Modified from Yamaguchi et al., J Electron Microsc 60, 337-351, 2011)

Unexpectedly, there was little variation in proportions of each organelle among cells (Fig. 3). Membranes of S. cerevisiae can be classified into two groups according to their thickness. The first group had a thickness of 16–19 nm and included the plasma membrane, vacuolar membrane, membranes of autophagosomes, and multivesicular body. The second group had a thickness of 13–14 nm and included the outer and inner nuclear envelope, the ER/Golgi membranes, and the mitochondrial outer membrane.

Fig. 4. Three-dimensional reconstructions of S. cerevisiae Cell 1–6. Red, nucleus; Green, mitochondria; Blue, vacuoles; Purple, endoplasmic reticula/Golgi apparatus; Yellow points, small vesicles; Red points, virus-like particles. *, position where mother cell attached. (Reprinted from Yamaguchi et al., J Electron Microsc 60, 337-351, 2011)

Structome analysis led to the discovery of an unknown microorganism. In 2010, Dr. Yamaguchi and his colleagues collected samples in a deep sea off the coast of Japan and observed microorganisms by electron microscopy. They found a yeast-like microorganism that had a cell wall and bacteria-like endosymbionts, but had no true nucleus nor mitochondria. By structome analysis it became evident that this microorganism was not a true eukaryote nor a true prokaryote. They named this microorganism ‘Parakaryon myojinensis’. Details of this study will be described in another section.

The genome is the whole genetic information of an organism; the proteome is the whole protein information of a cell or organism. Similarly, the structome is the whole structural information of a cell at the electron microscopic level, an important concept for understanding cell function.

This study was supported by the Ministry of Education, Culture, Sports, Science and Technology of the Japanese Government (19570053).

For more information, read articles:

  1. Yamaguchi M, Namiki Y, Okada H, Mori Y, Furukawa H, Wang J, Ohkusu M, Kawamoto S: Structome of Saccharomyces cerevisiae determined by freeze-substitution and serial ultrathin sectioning electronmicroscopy. J Electron Microsc. 60: 321-335, 2011.
  2. Yamaguchi M, Chibana H: A method for obtaining serial ultrathin sections of microorganisms in transmission electron microscopy. J Visual Exp, (131), e56235, doi:10.3791/56235, 2018.

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