The Shape of Mitochondria and the Number of Mitochondrial Nucleoids During the Cell Cycle of Euglena Gracilis

The shape of mitochondria and the number of mitochondrial nucleoids during the cell cycle of Euglena gracilis

YASUKO HAYASHI and KATSUMI UEDA*

Biological Laboratory, Nara Women's University, 630 Nara, Japan

* Author for correspondence

Summary

The shape of mitochondria and the number of during cytokinesis. Nucleoids in the newly pro- mitochondrial nucleoids in Euglena cells were duced daughter cells increased in number as the examined throughout the cell cycle by fluorescence cells increased in size. The number of nucleoids microscopy. Both photoheterotrophic and hetero- reached double the initial value in cells at the stage trophic cells contained a network of mitochondria just prior to mitosis. The total length of the mito- that did not divide into fragments at any stage of the chondrial net was proportional to the cell volume. cell cycle. Mitochondrial nucleoids could be clearly detected in the mitochondria by staining with ethidium bromide and with DAPI. Half of the mito- Key words: cell cycle, Euglena, fluorescence microscopy, chondrial nucleoids entered each daughter cell mitochondria, mitochondrial nucleoid.

Introduction detail (Kuroiwa, 1982). Fusion of mitochondrial nucleoids has also been traced in a yeast, Saccharomyces Fluorescence microscopy can provide valuable infor- cerevisiae, by fluorescence microscopy (Miyakawa et al. mation about the shape and patterns of distribution of 1984). However, reports on analysis of the morphology of mitochondria in the cell (Johnson et al. 1980, 1981; mitochondria and mitochondrial nucleoids by fluor- Goldstein & Korczack, 1981; Kuroiwa et al. 1981a; escence microscopy are very few in number, because this Terasaki et al. 1986). It is a much more convenient method has been only recently developed. Little is known technique than electron microscopy for investigation of about the number of nucleoids in the large reticulated such aspects of the morphology of mitochondria. It is not mitochondrion that is frequently found in algal cells, or necessary to make serial sections or to construct three- about changes in the numbers of nucleoids during the cell dimensional figures as in the case of electron microscopy. cycle. This report describes the shape of mitochondria Using fluorescence microscopy, one can accurately assess and the changes in the numbers of mitochondrial nu- the morphology of mitochondria in cells by the thousands cleoids during the cell cycle of Euglena. if an appropriate fluorescent dye, which specifically combines with the mitochondria, is employed. The fluorescent dye DiOC6(3) (3,3'-dihexyloxacarbo- Materials and methods cyanine iodide) was chosen for detection of mitochondria by Johnson et al. (1981). Hatano & Ueda (1988) and Green and leuco strains of Euglena gracilis Z were used in this Noguchi & Ueda (1989) compared the fluorescent struc- investigation. The leuco strain was a mutant induced by ture in a solution of DiOC6(3) with the mitochondria ultraviolet (u.v.) irradiation in our laboratory. Cells were examined by electron microscopy, and they proved that cultured at 25 °C in a culture medium that contained 0-05% the fluorescent structure corresponds to the mitochondria KH2PO4, 0-1% citric acid, 0-1% sodium citrate, 0-02% in algal cells. MgSO4, 0-5% glucose, 0-5% polypeptone, and 10/Journal of Cell Science 93, 565-570 (1989) Printed in Great Britain (6) The Company of Biologists Limited 1989 565 rinse with culture medium, cells were mounted in culture medium and examined under a fluorescence microscope (Olym- pus Kogaku Co., BHS-RFA type) with a blue excitation filter. Photographs were taken on Kodak Tri-X film. For double staining of mitochondria and mitochondrial nucleoids, cells stained with DiOC6(3) were further treated for 5 min with 0-2% ethidium bromide, washed with culture medium, and mounted in culture medium. Photographs were taken on Fuji DX-400 film under the same conditions as used for single staining with DiOC6(3). For counting the number of mitochondrial nucleoids, photographs were taken through many different optical focal planes in the cells, which were treated with DAPI (4',6-diamidino-2- phenylindole) (Sigma St Louis, USA) in a 0-05% aqueous solution. Each nucleoid shown in the photographs was carefully checked to ascertain whether it was visible in other photographs taken at the upper or lower adjacent focal planes, to avoid counting the same nucleoids more than once. For digestion experiments of mitochondrial nucleoids, cells were partially broken by a homogenizer in l/l5M-phosphate buffer (pH6'5) containing 5 mM-MgC^ and 0-5 M-mannitol, and were treated for 30 min at 30°C with deoxyribonuclease I (Sigma, St Louis, USA) at a concentration of ISOOKunitz units ml"1. The mitochondrial nucleoids in the broken cells were detected with ethidium bromide before and after digestion with the nuclease. The measurements of the total lengths of mitochondria and of the cell volumes were carried out on the photographic figures. As the cells were considerably pressed between coverslips and slides, the cells assumed a discoid shape with uniform thickness, so that the cell volume was represented by the surface area of the cell. The total lengths of the mitochondria were also represented by twice the length of the mitochondria that were photographed near the upper surface of the cell, because near the opposite surface of the cell the distribution of a similar quantity of mitochondria was observed.

Results

Mitochondria during the cell cycle A network of mitochondria was clearly visualized under the fluorescence microscope after staining with the dye DiOC6(3). The network was apparent in the leuco Euglena (Fig. 1A-D) as well as in the green Euglena (Fig. 2A-D). The network of mitochondria was situated close to the cell surface, surrounding other organelles in the cell. There was only one network of mitochondria in Fig. 1. Mitochondria in leuco Euglena. A. Growing cell. An each cell. The distribution density of the mitochondria in arrow points to the thin strand of mitochondria at the nuclear the network was usually low in the region above the region. B. Cell with two daughter nuclei («) formed by nucleus; one or two thinner strands of mitochondria were nuclear division. C. Dividing cell. D. Young cell soon after frequently seen in this region (see arrow in Fig. 1A). The cytokinesis. /", reservoir. X1400. network of mitochondria was also less dense in the region of the reservoir (see r in Fig. 1A). stages, which may result from the partial cutting of the The volume of the cell gradually increased during the networks. The cleavage of the cells by constriction growing phase and reached a maximum at the initial stage inevitably resulted in cutting of the mitochondrial of mitosis. Fig. IB shows the network of mitochondria at networks, so that the number of terminals in the networks the mitotic telophase, by which time two nuclei have been increased. Small cells, which were assumed to be those at produced. In Fig. IB, the mitochondrial network was stages soon after cytokinesis, also contained a network of absent over the nuclei and the reservoir. mitochondria (Fig. ID). A cleavage by constriction proceeded from the anterior The green Euglena emitted primary fluorescence from part to the posterior part of the cell after mitosis the chloroplasts. However, strong fluorescence from the (Fig. 1C). The networks of mitochondria were main- mitochondria, which were treated with DiOC6(3), en^ tained during the cleavage of the cells. The networks had abled us to trace the mitochondrial network accurately, more rod-shaped terminals than those found at previous provided that the primary fluorescence from chlorophylls

566 Y. Havashi and K. Ueda Mitochondria in green Euglena. A. Fully grown cell, in mitosis. C. Cell with two daughter nuclei after D. Dividing cell. /;, nucleus. X140Q,

Cell double-stained with DiOC6(3) and ethidium e. The network of mitochondria is colored green, point to the mitoehondrial nucleoids. n, nucleus. in the chloroplasts was blocked with a green filter. The their size (Fig. 4G). All the nucleoids in the daughter cell behavior and the appearance of mitochondria in the green are shown in Fig. 5D and 139 nucleoids can be counted. Euglena throughout the cell cycle were the same as those The total length of the mitochondrial net has been in the leuco Euglena (Fig. 2A-D). plotted against the cell volume in Fig. 6. The total length appears to be nearly proportional to the cell volume. Mitochondrial nucleoids Small cells, which probably were cells soon after cell Mitochondrial nucleoids could be visualized after the division, contained about 800jum of mitochondria, and treatment of cells with DAPI or ethidium bromide, the large cells contained about 1600 jitm. The number of under a fluorescence microscope. Fig. 3 is a fluorescence mitochondrial nucleoids was also proportional to the cell micrograph of a leuco Euglena double-stained with volume, but not as strictly proportional as the mitochon- DiOC6(3) and ethidium bromide. The nucleus strongly drial length. Small cells contained about 150 nucleoids, emitted a vermilion fluorescence (see n in Fig. 3). and large ones about 350. Mitochondria emitted a green fluorescence. There were many small granules in the network of mitochondria (see arrows in Fig. 3). These granules appeared to be yellow, Discussion probably as a result of the combination of their original vermilion and mitochondrial green. They lost their Early studies demonstrated that Euglena contained many fluorescence after treatment with DNase, indicating that ovoid or rod-shaped mitochondria, by the examination of they contain DNA. Accordingly, the granules were single random sections of the cell under the electron identified as the mitochondrial nucleoids. As shown in microscope (Gibbs, 1960; Malkoff & Buetow, 1964). Fig. 3, small fluorescent granules were not detected Reticular mitochondria were observed by Leedale & outside the mitochondrial network. This may imply that Buetow (1970) in living Euglena gracilis. Next, an the leuco strain used in this investigation contains no interpretation was proposed that the mitochondria are detectable plastid nucleoids. reticular in non-dividing cells and are rod-shaped in The leuco Euglena was used to examine the numerical dividing cells. The rod-shaped mitochondria grow and changes in the mitochondrial nucleoids during the cell fuse with each other to form a mitochondrial network cycle, in order to avoid misidentification of chloroplast after cell division (Calvayrac et al. 1972, 1974; Osafune

Mitochondria and mitochondrial nucleoids 567 Fig. 4. Mitochondrial nucleoids stained with DAPI. A. Growing cell. B-D. Three chosen photos suggesting the division of nucleoids. B. Elongated nucleoids. C. Pair of nucleoids positioned close to one another. D. Pair of nucleoids. E. Cell with two daughter nuclei. F. Dividing cell. G. Young cell soon after cytokinesis, a, assimilation product; n, nucleus. A, E-G, X1900. B-D, X7700.

568 Y. Hayashi and K. Ueda There have been several reports on the timing of synthesis of mitochondrial DNA during the cell cycles of various organisms. The synthesis of mitochondrial DNA in P. polycephalum occurs at a different time to that of nuclear DNA synthesis (Kuroiwa et al. 1978). Mitochon- dria in Tetrahymena pyriformis have been reported to incorporate thymidine at all stages of the cell cycle independently of nuclear DNA synthesis (Parsons & Rustad, 1968). By contrast, both the mitochondrial and nuclear DNA in Euglena appeared to be synthesized at the beginning of and during the first phase of cell division from an examination of incorporation of tritiated adenine (Calvayrac et al. 1972). In the present investigation, we did not find any specific figures of mitochondrial nucleoids that suggested the synthesis of mitochondrial DNA at the beginning of nuclear division in Euglena. As shown in Fig. 6, the number of mitochondrial nucleoids increased as the cell increased in volume after cytokinesis. If the amount of mitochondrial DNA is proportional to the number of nucleoids, then the mitochondrial DNA is probably synthesized continuously during the non-dividing, growing phase.

References

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570 Y. Hayashi and K. Ueda