three-dimensional behaviour of mitochondria during...

J. Cell Sci. 73, 207-220 (1985) 207Printed in Great Britain © The Company of Biologists Limited 1985

THREE-DIMENSIONAL BEHAVIOUR OF

MITOCHONDRIA DURING CELL DIVISION AND GERM

TUBE FORMATION IN THE DIMORPHIC YEAST

CANDIDA ALBICANS

KENJI TANAKA, TOSHIO KANBELaboratory of Medical Mycology, Research Institute for Disease Mechanism and Control,Nagoya University School of Medicine, Nagoya 466, Japan

AND TSUNEYOSHI KUROIWADepartment of Cell Biology, National Institute for Basic Biology, Okazaki 444, Japan

SUMMARY

This study was done to correlate mitochondrial behaviour with nuclear behaviour and celldivision as well as with the germ tube formation in the dimorphic yeast Candida albicans. Three-dimensional reconstruction of electron micrographs of serially sectioned cells of the three strainswas used to determine the morphological and quantitative relationships between the structures.The results suggested that at the time of entry into the bud a few mitochondria fused into a singlegiant one, which fragmented during mitosis and resumed a single giant form before cytokinesis,and was then partitioned into two parts. This tendency was also shown during germ tubeformation. Quantitative analysis has established that growth of organelles such as the nucleus andmitochondria closely followed total cell growth, the ratio of organelle volume to total cell volumebeing held relatively constant.

INTRODUCTION

Mitochondria proliferate not by de novo synthesis, but by the growth and divisionof the pre-existing organelles, keeping pace with nuclear and cell division. Duringvegetative growth of a yeast cell, it is commonly observed by both light and electronmicroscopy that portions of mitochondria migrate into the emerging bud and arepartitioned among daughter cells at cytokinesis by septum formation (Matile, Moor& Robinow, 1969).

Meanwhile, it is generally accepted that the numerous profiles of mitochondria inan electron micrograph represent cross-sections of a few branched structures in acell, and a number of observations have been published on the presence of a singlebranched mitochondrion, shown by three-dimensional reconstruction from serialsections in a wide variety of unicellular organisms such as yeast cells. In the bakers'yeast Saccharomyces cerevisiae, Hoffman & Avers (1973) first demonstrated the

Key words: mitochondria, Candida albicans, three-dimensional reconstruction, cell division,germ tube.

208 K. Tanaka, T. Kanbe and T. Kutviwa

presence of a single highly branched giant mitochondrion per cell by construction ofthree-dimensional models from consecutive sections of entire cells. However, sub-sequent studies have shown that, as conclusively stated by Stevens (1981) in herreview, yeast mitochondria are not static but dynamic, their number and structureundergoing extensive modification in accordance with the changes in the life cycleand the physiological state.

During the course of our studies on the three-dimensional reconstruction ofmitotic division in the imperfect yeast Candida, we frequently encountered theappearance of giant mitochondria, and decided to study the behaviour of theorganelles during cell division as well as germ tube formation in the dimorphic yeastCandida albicans. Ultrastructural changes were investigated not only qualitativelybut quantitatively by serial-section electron microscopy and three-dimensionalreconstruction. This paper presents the observations that fusion and division ofmitochondria are found in association with mitotic division of the nucleus.

MATERIALS AND METHODS

Organisms and cultures

Three strains of Candida albicans, NUM43, NUM678 and IAM4966, were used. StrainsNUM43 and NUM678 are clinical isolates maintained in our laboratory. Strain IAM4966 wasobtained from the Institute of Applied Microbiology, the University of Tokyo. Candida cellsmaintained on Sabouraud dextrose agar were inoculated for preculture into PGY broth (peptone,1 %/glucose, 2%/yeast extract, 0-5 % in distilled water) and incubated overnight with shaking at26°C. A 1-ml sample of the preculture was spread on a PGY agar plate, which was incubated forabout 6h to prepare the rapidly growing cells. Cells were harvested by flooding the fixative ontothe surface of the agar plate.

For the preparation of cells with germ tubes, Candida NUM678 cells at late logarithmic phaseof culture in PGY broth were harvested by centrif ugation and starved for 4 h by shaking in distilledwater at 37°C. The cells were then transferred into the germination medium containing S min-jV-acetyl-D-glucosamine (GlcNAc) in 25mM-HEPES (hydroxyethylpiperazine-yV-2-ethenesul-phonic acid) buffer adjusted to pH6'8 at a concentration of 2X107 cells/ml (Simonetti, Strippoli&Cassone, 1974). The cell suspension was incubated with shaking at 37°C and checked periodic-ally for germ tube emergence, by phase-contrast microscopy. Samples were taken at intervals andfixed for microscopy.

Electron microscopy

The fixatives used were 2-5% glutaraldehyde in 0-1 M-cacodylate buffer or 3 % glutaraldehydein ( H M - P I P E S (piperazine-N'-bis(2-ethanesulphonic acid)) buffer (pH6-94) containing 1 mM-MgClz and 1 mM-EGTA (ethylene glycol bis(2-aminoethylether)-tetraacetic acid). Both fixativesgave the same results. Subsequent procedures followed the method described by Tanaka & Hirata(1982). Briefly, the fixed cells were treated with Zymolyase 60000 (Kirin Brewery Co., Ltd)(0-1 mg/ml) in the same buffer at 30°C to digest the cell wall. After postfixation in 2 % OsO* andsoaking in 0-5 % aqueous uranyl acetate, cells were embedded in agar blocks and dehydratedthrough a series of ethanol and acetone before embedding in Spurr's resin. Serial sections wereobtained with a Dupont diamond knife on a Reichert Ultracut 0mU4, mounted on Formvar-coated single-slot grids, and stained with uranyl acetate and lead citrate. Sections were viewed witheither a JEOL 100CX or a Hitachi H-500 electron microscope at 100 kV.

3-D reconstruction of mitochondria in Candida 209

Three-dimensional reconstructions and the quantitative analysis of the organelles

All the sections of an entire cell were photographed at the appropriate magnifications between8300 and 15 000 and the outlines of cell and organelle profiles were drawn on a tracing paper byprojecting the negative at a precisely determined magnification. The volumes of the whole cell,nucleus, mitochondria and vacuole were calculated on an Image Analyzer Digigrammer/Model Gsystem (Mutoh Kogyo Co., Ltd) by tracing the profile of each structure on the digitizing table.Section thickness was calculated by determining the number of sections that covered a sphericalbody of a given diameter, and was found to be in the range from 75 to 90 nm. By superimposingthe tracings of a single cell it was possible to follow the continuity and topological relationship oforganelles and thus to estimate the shape and number of each organelle. Several series of sectionswere used for reconstructing three-dimensional models of mitochondria with balsa wood. Theywere made by tracing the outlines of mitochondrial profile from the master tracings onto thin balsawood, equal in thickness to the ultrathin sections multiplied by the magnification of the negative.All fragments were numbered and, after cutting up, were stacked up. Three-dimensionalreconstruction was also attempted by computer-aided graphics. Mitochondrial profiles wereentered in an Image Analyzer MOP Videoplan (KONTRON). The reconstructed profile wasprojected on a terminal display and printed out. A pair of projections turned at 6° to each otherwere printed to obtain a stereo pair, which gave a three-dimensional appearance of the structurewith a stereo viewer.

RESULTS

Three-dimensional reconstruction of mitochondria in dividing cells

Profiles of mitochondria appeared in the electron micrographs as oval toelongated, sinuous or branched rods. The width of the mitochondrion was about0-3-0-4/im, and did not vary much among the three strains studied. We studied aseries of electron micrographs from nine cells of strain NUM43, five cells of strainNUM678 and five cells of strain IAM4966 at the stage of exponential growth.NUM43 cells were small and ovoid, around 3-0jxra in diameter. NUM678 werelarger oval cells than NUM43, 3-5-4-0/xm in diameter, while IAM4966 wereheterogeneous in size and shape. Before describing the mitochondrial changes inrelation to cell division, a few examples of three-dimensional reconstruction arepresented in Figs 1-3. Fig. 1 shows a model of a single mitochondrion reconstructedwith balsa wood from 37 sections of a dividing cell of NUM43. We can see the modelfrom various directions in order to realize directly the three-dimensional structure.A single mitochondrion consisted of two leaves of reticulum, both of which, thoughnot strictly, took their symmetry in relation to the constricted point of the cellisthmus. A highly branched, single large mitochondrion in a NUM678 cell ispresented also in the balsa wood model in Fig. 2. The structure of the mitochondrialsystem at cell division in a IAM4966 cell is represented in Fig. 3. A stereo pair ofcomputer-aided reconstructions did not give us the exact number of mitochondria,but the topological relationship of the whole system of mitochondria was evident.The cell in Fig. 3 has 10 mitochondria.

All the observations that we analysed three-dimensionally through serial sectionson 19 cells from the three strains are summarized schematically in Figs 4-6. Cellsare presented in the presumptive order of progression of mitosis, which is indicated

K. Tanaka, T. Kanbe and T. Kuroiwa

1A

>r \

2A —Figs 1 and 2

B


by the sites of nucleus-associated organelles (NAO) (Girbardt & Hadrich, 1975) orthe poles of the mitotic spindle. There is a similarity in the sequence of mitochon-drial topology between strains NUM43 and NUM678. A few mitochondria werefound in an interphase cell of NUM43 (Fig. 4A), while only one was found in thecells with a bud (Figs 4B,C,D and 5A). In the budding cells with a mitotic spindle,several mitochondria are distributed between the mother and the bud cells (Figs 4E,5B,C,D). At the end of nuclear migration, the mitochondria fused into a single giantone, forming figure-of-eight shape nearly symmetrical with the point of the cellisthmus (Figs 4F, 5E), where the mitochondrion divided (Fig. 4G). Completion ofcytokinesis resulted in two daughter cells, each carrying a single mitochondrion(Fig. 4H), which again could undergo division (Fig. 4i). Strain IAM4966 had moremitochondria than the other strains described above (Fig. 6A), but the cells with a

,4 V

6

B

V.7

Fig. 3. Mitochondria in cell E of 1AM4966 shown in Fig. 6. A. A section from 45 serialsections (X6200); B, a stereo pair of computer-aided reconstructions.

Fig. 1. A single mitochondrion in cell F of NUM43 shown in Fig. 4. A. A section froma series of 37 serial sections (X 14 600); B and c are the surface and side views of a balsawood model of the mitochondrion.

Fig. 2. Three-dimensional representation in balsa wood of a single branched mitochon-drion reconstructed from 54 serial sections of cell E of NUM678 shown in Fig. 5. A andB are surface and side views of the model, respectively.

212 K. Tanaka, T. Kanbe and T. Kuroiwa

Fig. 4. Schematic drawings of mitochondria in relation to the nucleus in NUM43 cellsanalysed by three-dimensional reconstruction from serial sections of entire cells. Filledareas are mitochondria, and hatched areas show the nucleus, in which arrowheadsindicate the sites of NAOs or spindle pole bodies.

Figs 5,6. The same as in Fig. 4, but cells of NUM678 are represented in Fig. 5 and thoseof IAM4966 in Fig. 6.


mitotic nucleus had a smaller number of elongated structures (Fig. 6B,C,D). Thissuggested that mitochondrial fusion might have occurred before and/or duringmitosis. Elongated mitochondria were again fragmented into smaller ones, whichseemed to be distributed equally into the two daughter cells before nuclear separ-ation occurred (Fig. 6E).

Quantitative analysis of cell organelles

All the cells drawn schematically in Figs 4—6 were analysed for cell, nuclear andmitochondrial volumes, their ratios compared with the total cell volume, and thevacuolar volumes were analysed by an image analyser as described in Materials andMethods. The results are shown in Table 1. Total cell volumes represent changesduring the cell.division cycle and the differences in cell size between the strains. InNUM678, smaller volumes were obtained in cells A and B compared to the otherthree cells. This might be due to the differences in ploidy among the cells studied,because a culture of NUM678 contained both diploid and tetraploid cells asdetermined by fluorescent microphotometry (Suzuki, personal communication). Aremarkable finding was that cell components such as nucleus and mitochondriamaintained relatively constant proportions of the total cell volume throughout theprogressive changes leading to cell division in NUM43 or through the mitotic stagesin the other two strains. The nucleus and mitochondria increased and almostdoubled their volume before cytokinesis, after which two daughter cells wereformed, each carrying an approximately equal amount of cell organelles, as far asorganellar topology was concerned.

Mitochondrial behaviour during germ tube formation in NUM678

Growing cells starved in distilled water and incubated in HEPES buffer contain-ing GlcNAc produced germ tubes and all the yeast cells were transformed intohyphal cells within 2h. We also studied the topological relationship between theorganelles during germ tube formation. A mitotic cell with an emerging germ tubecontained seven mitochondria (Fig. 7), one of which was moving into the germ tube.Several mitochondria were fused into a single giant one as the germ tube elongated(Figs 8, 9), and then split into a few smaller organelles after nuclear migration. Aschematic representation depicting changes in mitochondria in relation to nucleardivision that occurred concurrently with germ tube emergence is shown in Fig. 10.Organellar volumes and their ratios to total cell volumes analysed quantitativelyduring germ tube elongation again showed results comparable to those obtainedduring cell division: nuclear and mitochondrial volumes increased but their ratios tocell volume remained relatively constant (Table 2). Changes in the vacuolar volumeand its relative proportion in the cell seemed to be dependent on the stage of the cellcycle.


B

8A

Figs 7-10. C. albicans NUM678 cell with a germ tube.Fig. 7. A system of seven mitochondria in cell A shown in Fig. 10 with an emerging

germ tube. A. A section from a series of 73 sections. X13 200. B,C. Views of a balsawood model of a mitochondrial system.

Fig. 8. Cell B shown in Fig. 10. A. A section from a series of 33 sections. X12 600.B. A stereo pair of a single giant mitochondrion entering into the germ tube.

216 K. Tanaka, T. Kanbe and T. Kurvtwa

Fig. 9


Fig. 10. Schematic drawings of mitochondria in relation to nuclear division andmigration in germ tube formation in NUM678, analysed by reconstruction from serialsections of whole cells. Filled areas are mitochondria and hatched.areas are nuclei inwhich the sites of NAOs are indicated by arrowheads.

DISCUSSION

The present investigation has elucidated the fine structure of mitochondrialbehaviour during mitosis and germ tube formation in the dimorphic yeastC. albicans. In strains NUM43 and NUM678, at the time of entry into the bud a fewmitochondria fused into a single giant organelle, which fragmented during mitosisand re-formed a single one with a figure-of-eight-shaped structure surrounding themigrating nucleus before cytokinesis. Then the structure divided into two parts,each surrounding a nucleus in the daughter cell (Figs 4, 5). The behaviour of strainIAM4966 was not as simple as that of the other two strains, but the entry of a giantmitochondrion into the bud and the equal distribution of mitochondria between the

Fig. 9. Cell C shown in Fig. 10 with an elongated germ tube. A. A section from 57serial sections. X9700. B,C. Surface and side views, respectively, of the model re-constructed with balsa wood.


two daughter cells were observed (Fig. 6). A giant structure was also developedduring germ tube formation (Fig. 10).

The importance and the problems of the analysis of mitochondrial structure byserial thin-sectioning and electron microscopy were discussed by Stevens (1977),who concluded that the number and form of mitochondria in Saccharomycescerevisiae are not related to the stage in the cell cycle but to the phase of growth.However, our studies have demonstrated that mitochondria in C. albicans under-went changes of fusion and division in association with mitosis in the cell cycle.Taylor & Wells (1979) also stated that mitochondrial division was not strictlycorrelated with nuclear division in an imperfect yeast Bullera alba. Their recon-structed mitochondria were not presented in relation to the mitotic nucleus, but theobservations of a large mitochondrion in premitotic cells and one to three in mitoticones are not necessarily in conflict with our observations.

The actual sequence of mitochondrial behaviour during the cell cycle could bedetermined by an analysis of a synchronous culture, but it would be impossible toanalyse sufficient cells by serial-section electron microscopy. In this paper, theprogression of the cell cycle in Candida cells was correlated with the extent of budemergence and spindle development in the nucleus. The latter, in turn, wasestimated by the distance between the two opposite nucleus-associated organelles.These morphological changes were in good correlation with a quantitative analysis oforganellar volumes. It was well established that mitochondria as well as the nucleusincreased their volume as the cell size increased, the ratio of organelle volume to totalcell volume remaining almost constant. Mitochondrial fusion and division did notseem to be dependent on the growth or increase in volume of a whole system ofmitochondria but on the stage of the cell cycle.

The average percentage of cell volume occupied by mitochondria was in the rangefrom 6 to 13 % according to the strains used. The number and volume of mitochon-dria in a cell are reported to be dependent on physiological conditions (Stevens,1977; Taylor & Wells, 1979). Although we did not compare cells grown underdifferent conditions, our results are similar to the data reported for other yeast cells(Taylor & Wells, 1979; Grimes, Mahler & Perlman, 1974; Keddie & Barajas, 1969).In addition, we have demonstrated that the number and volume of organelles aredifferent among the strains used.

We are indebted to Mrs Shigeko Yagi for her enduring work on three-dimensional reconstruc-tion of the structure and quantitative analysis of the data. This work was partly supported bygrants from the Ministry of Education, Science and Culture of Japan and the Toray ScienceFoundation. This study was carried out under the NIBB Cooperative Research Program (83-8).

REFERENCES

GIRBARDT, M. & HADRICH, H. (1975). Ultrastruktur des Pilzkernes III . Genese des Kern-assoziierten Organelles (NAO). Z. allg. Mikrobiot. 15, 157-173.

GRIMES, G. W., MAHLER, H. R. & PERLMAN, P. S. (1974). Nuclear gene dosage effects onmitochondrial mass and DNA.J . Cell Biol. 61, 565-574.

220 K. Tanaka, T. Kanbe and T. Kurotwa

HOFFMAN, H. P. & AVERS, C. J. (1973). Mitochondrion of yeast: Ultrastructural evidence for onegiant, branched organelle per cell. Science 181, 749-751.

KEDDIE, F. M. & BARAJAS, L. (1969). Three dimensional reconstruction of Pityrosporum yeastcells based on serial section electron microscopy. J . Ultrastruct. Res. 29, 260-275.

MATILE, PH. , MOOR, H. & ROBINOW, C. F. (1969). Yeast cytology. In The Yeast, vol. 1 (ed. A. H.Rose & J. S. Harrison), pp. 219-302. London: Academic Press.

SIMONETTI, N., STRIPPOLI, V. & CASSONE, A. (1974). Yeast-mycelial conversion induced by N-acetyl-D-glucosamine in Candida albicans. Nature, Land. 250, 344—346.

STEVENS, B. (1977). Variation in number and volume of mitochondria in yeast according togrowth conditions. A study based on serial sectioning and computer graphics reconstruction.Biol. Cell. 28, 37-56.

STEVENS, B. (1981). Mitochondrial structure. In The Molecular Biology of the Yeast Saccharo-myces cerevisiae. Life Cycle and Inheritance (ed. J. N. Strathern, E. W. Jones & J. R. Broach),pp. 471-504. New York: Cold Spring Harbor Laboratory.

TANAKA, K. & HIRATA, A. (1982). Ascospore development in the fission yeasts Schizosaccharo-myces pombe and S. japonicus. J. Cell Sci. 56, 263-279.

TAYLOR, J. W. & WELLS, K. (1979). The mitochondrion in mitotic and starved cells of Bulleraalba. Expl Mycol. 3, 16-27.

(Received 25 June 1984 -Accepted 2 August 1984)

three-dimensional behaviour of mitochondria during...

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