STUDIES OF BUDDING AND CELL WALL STRUCTURE OF YEAST

ELECTRON MICROSCOPY OF THIN SECTIONS'

HILDA D. AGAR AND H. C. DOUGLAS

Department of Microbiology, University of Washington School of Medicine, Seattle, Washington

Received for publication April 15, 1955

Observations of intact yeast cells with theelectron microscope have yielded little informa-tion on cellular structure because of the im-penetrability of the cells to the electron beam.The development of techniques for the prepara-tion of ultra-thin sections promises to obviatethis difficulty, and several papers have alreadyappeared in which this approach has been ap-plied in studies of the structure of microorganisms(Chapman and Hillier, 1953; Birch-Andersonet al., 1953; Wolken and Palade, 1953; Gustafsonet al., 1954).

In this paper we describe details of the buddingprocess and the structure of the cell wall ofSaccharomyces cerevisiae as observed in thinsections of intact cells and purified cell walls.

METHODS

A tetraploid S. cerevisiae strain IF-5 was usedfor the major part of this study, although somework was also done with a haploid strain. Thetetraploid strain was chosen because of its largersize and because it had been studied cytologicallyand genetically by other workers in this depart-ment (Hawthorne, 1955). Vigorously buddingcultures in the logarithmic phase were used.These were obtained by transferring cells to tubesof broth (1 per cent yeast extract, 1 per centpeptone, 2 per cent glucose) from an overnightculture and incubating on a shaker at roomtemperature for 4 to 6 hours. The cultures werecentrifuged and the supernatant discarded andreplaced by an osmium tetroxide fixative solu-tion. The 1.0 per cent buffered fixative of Palade(1952) and the fixative of Chapman and Hillier(1953) gave equally good results if the fixationtime was prolonged to 16 to 20 hours. Osmiumconcentrations below 1 per cent and shorter

' Supported in part by a grant from the Stateof Washington fund for Biological and MedicalResearch.

fixation times resulted in swelling, vacuolization,and disruption of the cells.

Following fixation, the cells were dehydratedby passage through a graded series of ethanolconcentrations, impregnated with methacrylatemonomer, and polymerized in gelatin capsulesas described by Newman et al. (1949). A mixtureof 4 parts N-butyl methacrylate and 1 partmethyl methacrylate monomer gave blocks ofthe most satisfactory hardness for thin sectioning.

Sections were cut with a modified SpencerRotary Microtome (advanced by thermal ex-pansion) and more recently with a Porter Blummicrotome.2 Glass knives prepared as describedby Latta and Hartmann (1950) were used withboth microtomes. The sections, as cut, werefloated off on the surface of a 50 per cent acetone-water solution behind the knife edge and pickedup on formvar coated specimen grids. The sec-tions were drained on filter paper, air dried, andselected for suitable thinness by their inter-ference colors when viewed under a directillumination microscope at 80 X. The sectionswere studied with an RCA model EMU-2belectron microscope equipped with a self-biasedgun and a limiting aperture in the objectivepole piece to improve contrast. Electron micro-graphs were taken at initial magnifications of3,800 X to 9,000 x with further photographicenlargement as indicated.

Cell walls were prepared by disrupting yeastsuspensions in a Mickel tissue disintegrator,followed by differential centrifugation and wash-ing. The cell walls were then fixed, dehydrated,and embedded in methacrylate as describedabove.

RESULTS AND DISCUSSION

A longitudinal section through a mother cellwith two buds (A and B) at different stages of

2 Manufactured by Ivan Sorvall Inc., P.O. Box230, Pearl Street, Norwalk, Conn.

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Figure 1. Electron micrograph of a longitudinal section through a budding yeast cell. 14,000 X. A,bud with cytoplasm continuous with that of mother cell; B, mature bud with developing cross wall be-tween mother and daughter cell; C, bud scar; D, extension of cell wall material into cytoplasm.

development is shown in figure 1. The cytoplasmof bud A is still continuous with that of themother cell, while in bud B the cytoplasmic con-tinuity no longer exists. The extension of cellwall material into the cytoplasm of the mothercell and bud B is evident at D and, as will be

pointed out below, this phenomenon appears tobe characteristic of the later stages of the buddingprocess. A well-defined bud scar is present at C.In whole mounts of isolated cell walls, a bud scarappears as a circular, raised rim on the surfaceof the cell wall (Northcote and Horne, 1952;

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FigJiiie 7. Douible cross wsall eompletely formeJl s;howiii- the geometric fit of b)ud scar and b)irth scar.38,00() X.

Figiii-e 8. Siingle yeas;t (cll NAith b>irth seatr at upIper iiig,ht, alwl boud scarl at lowser left. 29,000 X.

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BUDDING AND CELL WALL STRUCTURE OF YEAST

Figure 9. Longitudinal section, shows lamellae of inner layer of cell wall.Figure 10. Longitudinal section, shows lamellae of the bud scar.

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Houwink and Kreger, 1953). The section throughthe scar at C shows the elevated edge of the rimand the enclosed, rounded plug of cell wall ma-terial which forms the surface of the scar. Thediscontinuity in wall structure at the point ofcontact of the plug and rim of the bud scar is alsoapparent.Very little structural detail is evident in the

cytoplasm of the cells in figure 1 or the succeedingfigures. This is due to difficulties in sectioningwhich, in our experience, appear to be char-acteristic of yeast. In many cases the cytoplasmappears to be corrugated and somewhat com-pressed. This "wash-boarding," caused ap-parently by internal chatter during cutting,makes difficult a study of structural detail in the

.1Figure 11. Electron micrograph of a longitudinal section through a yeast cell showing the electron

dense fibrils lining inner surface of cell wall. 29,000 X.

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BUDDING AND CELL WALL STRUCTURE OF YEAST

Fig. 12. Electron micrographs of sectioned cell walls showing laminated structure of the wall. 28,000 X.

cytoplasm. None of our sections have shownstructures which could be identified as nuclei,vacuoles, or cytoplasmic granules, although theirpresence in the cells of strain IF-5 can easily bedemonstrated by other techniques.

Figures 2 to 8 represent cells in different stagesof bud formation. An early stage is seen in figure2, where the bud is represented by a small bulgein the wall of the mother cell. A somewhat laterstage is presented in figure 3. In figure 4 the budis quite large but the cytoplasm of the two cellsis still continuous. The formation of the wallwhich separates the cells is shown in figures 5and 6. In both figures there appears the char-acteristic extension of the cell wall material intothe cytoplasm which was noted previously infigure 1. While it is difficult to interpret thesignificance of this phenomenon it appears to usto represent a stage of active synthesis of the cellwall material, which eventually forms the convexand concave plugs of cell wall sealing the con-stricted opening between the cells. A cleavageline can be seen in the new cell wall material infigure 6, while in figure 7 cleavage appears to becomplete and the geometric fit of the concave budscar and the convex birth scar is clearly demon-strated. Figure 8 shows a single cell with a con-cave birth scar at one pole and a convex bud scarat the other.

The precise manner in which the cell wallseparating mother and daughter cells is laid downis not known, but our micrographs indicate thatits formation might well resemble the pattern ofcentripetal extension of the external cell wall thatoccurs in bacteria (Knaysi, 1941; Chapman andHillier, 1953), with the essential difference beingthat new cell wall material may originate over amuch wider area. This is suggested by the ap-pearance of figures 5 and 6 described above, andalso by the structure of the bud scars. The plugof cell wall material of the bud scar appears tobe attached to the inner side of the external cellwall over a considerable area (figures 1 and 10),and it may well be that the cell wall material ofthe plug originated over the entire area whereplug and external wall are contiguous.With regard to the structure of bud and birth

scars, our observations are in agreement withthose of Barton (1950), who first pointed out thatthe two could be distinguished by their respectiveconvex and concave appearances. This was ques-tioned, however, by Bartholomew and Mittwer(1953), who found no difference in structure ofbud and birth scars of cells that had been treatedwith ultraviolet light to make them more trans-parent to the electron beam.The cell wall of S. cerevisiae is known to be a

complex structure which consists principally of

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glucan and mannan, and smaller amotnts ofprotein, lipid and chitin (Northcote and Horne,1952; Houwink and Kreger, 1953). Northcoteand Horne also presented chemical and micro-scopic evidence which indicated that the cell wallconsists of at least 2 distinct layers, one of whichis composed exclusively of glucan. Their observa-tions also suggested a role for lipids as cementingsubstance in the laminated cell wall, for whenpurified cell walls were extracted with lipidsolvents separation of the wall into two mem-branes was observed.Our observations are in agreement with the

conclusion of Northcote and Horne that the cellwall of Saccharomyces consists of distinct layers.This is evident in the section shown in figure 9.The outer layer of the cell wall consists of electrondense material, about 0.05 ,u in average thickness,which has a somewhat filamentous appearancein most sections. Beneath this is a layer of lesserelectron density which has an average thickness ofabout 0.2 ,u, and which is subdivided by thin,electron dense lamellae that are particularlyprominent at or near the bud scars (figure 10).Lining this layer is what appears to be a networkof electron-dense fibrils which are quite sinuatein form (figure 11). Whether these structures arepart of the cell wall or the cytoplasm is difficultto say, but according to Houwink and Kreger(1953) the innerside of the cell wall of S. cerevisiacis lined with a network of fibrils.When sections of isolated cell walls were ob-

served, the laminated nature of the cell wall wasquite evident, particularly in areas near scarswhere the cell wall had separated. This is shownin figure 12, where the wall has separated intothree distinct layers. In none of the cell wallpreparations, however, was there any suggestionof the presence of the electron dense outer layerwhich was so prominent in the sections of wholecells. It is possible that this layer is lost duringthe manipulations required for the preparationof the cell walls, although extensive washing ofwhole cells both before and after fixation did notalter the appearance of this outer layer.Study of this yeast is being continued with the

hope of obtaining sections satisfactory for theobservation of cytoplasmic detail.

SUMMARY

Thin sections of yeast cells and "purified"yeast cell walls have been prepared and examined

with the electron microscope. Some of the stagesof the budding process have been described.The yeast cell wall consists of several distinct

layers. Observations of cell wall structure madeon sections of whole cells and isolated cell wallswere in general agreement, with the exceptionthat an outer layer of electron dense materialwas consistently present in whole cells but not inisolated cell walls.

Details of the internal structure of the cellswere obscured due to difficulties of fixation andsectioning.

REFERENCESBARTHOLOMEW, J. W., AND MITTWER, T. 1953

Demonstration of yeast bud scars with theelectron microscope. J. Bacteriol., 65, 272-275.

BARTON, A. A. 1950 Some aspects of celldivision in Saccharomyces cerevisiae. J. Gen.Microbiol., 4, 84-86.

BIRCH-ANDERSON, A., MAAL0E, O., AND SJO-STRAND, F. S. 1953 High-resolution electronmicrographs of sections of E. coli. Biochem.et Biophys. Acta, 12, 395-400.

CHAPMAN, G. B., AND HILLIER, J. 1953 Electronmicroscopy of ultra-thin sections of bacteria.J. Bacteriol., 66, 362-373.

GUSTAFSON, P. V., AGAR, H. D., AND CRAMER,D. I. 1954 An electron microscope study ofToxoplasma. Am. J. Trop. Med. Hyg., 3,1008-1021.

HAWTHORNE, D. L. 1955 The use of linear ascifor chromosome mapping in Saccharomyces.Genetics, accepted for publication.

HOUWINK, A. L., AND KREGER, D. R. 1953Observations on the cell wall of yeasts.Antonie van Leeuwenhoek, 19, 1-24.

KNAYSI, G. 1941 Observations on the celldivision of some yeasts and bacteria. J.Bacteriol. 41, 141-154.

LATTA, H., AND HARTMANN, J. F. 1950 Use of aglass edge in thin sectioning for the electronmicroscope. Proc. Soc. Exptl. Biol. Med.,74, 436-439.

NEWMAN, S. B., BORYSKO, E., AND SWERDLOW, M.1949 Ultra-microtomy by a new method.J. Research Nat. Bur. Standards, 43, 183-199.

NORTHCOTE, D. H., AND HORNE, R. W. 1952Chemical composition and structure of theyeast cell wall. Biochem. J., 51, 232-236.

PALADE, G. E. 1952 A study of fixation forelectron microscopy. J. Exptl. Med., 95:285-298.

WOLKEN, J. J., AND PALADE, G. E. 1953 Anelectron microscope study of two flagellates.Chloroplast structure and variation. Ann.N. Y. Acad. Sci., 56, 873-889.

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