Observations of Nuclei in the Moving Mitochondria of Live Physarum

CELL STRUCTURE AND FUNCTION 6, 255-262 (1981) C by Japan Society for Cell Biology

Observations of Nuclei in the Moving Mitochondria of Live Physarum Cells by Means of a Double Fluorescent Staining Technique, Using Ethidium Bromide and Dimethylaminostyrylmethylpyridiniumiodine (DASPMI)

Tsuneyoshi Kuroiwa, Shigeyuki Kawano, Takahito Suzuki and Soryu Nishibayashi

Department of Cell Biology, National Institute for Basic Biology, Okazaki 444, Japan

ABSTRACT. Ethidium bromide has been shown to be useful as a probe to observe mitochondrial nuclei (mt-nuclei) in the moving mitochondria in live cells. In Physarum polycephalum, a double staining technique, using ethidium bromide and dimethylaminostyrylmethylpyridiniumiodine, detects both changes in mitochondrial distribution and orientation, and changes in the shape of the mt-nucleus during the mitochondrial division cycle. The results show that when dumbbell-shaped mitochondria in live cells divide to form daughter mitochondria, each mt-nucleus also divides, separating into the daughter mitochondria.

Physarum mitochondria contain an electron dense nucleus (mt-nucleus), which consists of large amounts of DNA (11), RNA (10) and proteins (11-13). Therefore, one mitochondrial division involves two main processes at least—mitochondrial nuclear division and mitochondriokinesis (division of the mitochondrial matrix and cristae) (15). In the live cells of many organisms including Physarum polycephalum, mitochondriokinesis has been observed by phase-contrast microscopy (3, 4) or Nomarski interference microscopy (9). Although mitochondriokinesis and mt-nuclear division in fixed cells have been studied respectively by acid fuchsin and HCl-thionine staining (13), much less attention has been paid to the structure of the mt-nucleus during the mitochondrial division in live cells. In addition, previous techniques were used for fixed cells and therefore did not show mt-nuclei in live cells. Mitochondria and chloroplasts contain only very small amounts of DNA. There- fore, fluorescent probes such as 4'6-diamidino-2-phenylindole (DAPI) and Hoechst 33258, which display high specificity for DNA in organelles, are extremely useful when studying mitochondrial and chloroplast nuclei (2, 7, 8, 18). Hajduk (7) used DAPI as a fluorescent probe to demonstrate the presence of kinetoplast DNA in live cells of normal and dyskinetoplastic strains of Trypanosoma equiperdum, a unicellular protozoan. However, Trypanosoma cells did not move as if the cells may have died and the survival times of the cells were not indicated. Use of DAPI and Hoechst 33258 raises the question : How long can the organisms live under the ultraviolet

Abbreviations used: mt-nucleus, mitochondrial nucleus; DAPI, 4'6-diamidino-2-phenylindole; DASPMI, dimethylaminostyrylmethylpyridiniumiodine.

255 256 T. Kuroiwa et al. irradiation of epifluorescenct microscopy? DAPI requires a wavelength of 350-nm (Fig. 1a), a level which would damage most live organisms. Alternatively, fluoro- chrome ethidium bromide, which can be excited by light with longer wave lengths, have proven useful when the mt-nucleus in live cells is to be observed (Fig. lb). The excitation spectrum of ethidium bromide peaks at 300-nm and 500-nm (16) whereas DAPI has a single excitation peak. The use of green light (500-nm) to excite ethidium bromide enables epifluorescenct microscopy of the mt-nuclei in moving mitochondria. The purpose of the present study is to examine the size and shape of these nuclei throughout the entire mitochondrial division cycle using the above method.

MATERIAL AND METHODS

Plasmodium and swarm cell cultures. Physarum polycephalum microplasmodia were

obtained by the method reviewed by Guttes and Guttes (6). They were approximetely 200 ƒÊm

in diameter and were grown in reciprocal shaking cultures. Mitotically synchronized plasmo-

dia were prepared by fusing the microplasmodia. Surface plasmodia obtained between the

first postfusion mitosis (MI) and the third postfusion mitosis (MIII) as well as microplas-

modia and swarm cells were used. Swarm cells were obtained from myxoamoebae grown with Escherichia coli K-12 according to the method of Suzuki and Kuroiwa (17). The first

synchronous nuclear division of surface plasmodia occurred 8 h after fusion, the second

division 18 h and the third division at 28 h, while semisynchronous mitochondrial division

occurred approximately 2 h after mitosis. Small explants, 0.5 to 1 mm in diameter, were harvested from the plasmodium 0 to 20 h later at one hour intervals after MI, and divided into two groups.

Techniques to stain moving mt-nuclei. To study the mitochondrial division cycle, explants of one group were fixed in a buffer-Sl (0.75 M sucrose, 1 mM EDTA, 0.6 mM spermidine,

0.05 % mercaptoethanol, 10 mM Tris-HCl at pH 7.6) for 5 min. The explants were placed in a drop of ethidium bromide solution (50 ƒÊg/ml) dissolved in the buffer-Sl and mounted with a cover slip to make a squashed preparation.

To observe the mt-nuclei in the moving mitochondria of live cells, a second group was placed on 1 % agar plate or on a glass slide for between 5 and 10 min before staining. The explants were put in the central area of the slide, surrounded by a vaseline-circular barrier, and a drop of ethidium bromide (50 ƒÊg/ml), or a mixture of both ethidium bromide (50 ƒÊg/ml) and DASPMI (500 µg/ml) dissolved either in the culture medium or in buffer-S (0.25 M sucrose, 1 mM EDTA, 0.6 mM spermidine, 0.05 % mercaptoethanol, 10 mM Tris-HCI at pH

7.6) was added to the samples. A cover glass was placed on the sample. DASPMI, which was first used by Bereiter-Hahn (1), was used to permit recognition of all of the mitochondria in the live cells.

Epifluorescent microscopy. Stained cells were examined with an Olympus BH fluorescent microscope equipped with a phase objective lens employing epifluorescent illumination at either 520 nm (ethidium bromide excitation) or 450 nm (DASPMI excitation). A combina- tion of both epifluorescent and transmission phase-contrast microscopy was used to detect the outlines of the mitochondria and the mt-nuclei simultaneously. Photographs were taken with Kodak Tri-X (ASA 400) or Fuji Neopan (ASA 400) film with exposure times of 20-50 sec. A Shimazu RF-520 double beam recording spectrofluorometer was used to measure the excitation spectra of ethidium bromide and DAPI dissolved in distilled water (10 µg/ml and

1 µg/ml respectively).

Video and magnetic tape recordings of mt-nuclei. The mt-nuclei in moving mitochondria were recorded by the night vision techniques of Gilkey et al. (5). Using the epifluorescent Mitochondrial Nuclei in Live Cells 257 microscope, a magnifiedimage of the object was focused on the cathode of a silicon intensifier target tube (Night Vision Camera, NVC-100). The output phosphor of the intensifier was viewed on a silicon intensifier target 4804 (Ikegami CTC 9000). The camera image was projected in real time on a television monitor (Ikegami PM-121T) and simultaneously recorded on magnetic tape (National NV 8030). Photographs for analysis and display of videotape playbacks were taken afterwards. Swarm cells or microplasmodia were irradiated under light of varying wavelengths with an epifluorescentilluminator through the UVFL 100 objective, and the mean survival times per 30 cells were determined.

RESULTS Fig. 1b shows the survival times of microplasmodia and swarm cells after irradiation using various wavelengths. The majority of swarm cells died within 20 sec after the onset of UV-irradiation .Multinucleated microplasmodia were more resistant than the swarm cells, but non survived longer than 5 min under continuous irradiation at 400-nm. Therefore, DAPI, which requires short wavelength UV excitation, could not be used as the fluorescent prove. On the other hand, wavelengths greater than 500-nm were entirely harmless (Fig. lb) and were used for these observation. The plasmodium cell cycle and the structure of fixed plasmodium mitochondria and of mt-nuclei were examined by phase contrast-fluorescent microscopy for their entire division cycle. Figs. 2a-c consist of phase contrast (Fig. 2a), fluorescent (Fig.

Fig. 1. a) Excitation spectra of ethidium bromide (-) and 4'6-diamidino-2-phenylindole (-). b) The effects of continuous excitation by means of various wavelengths on microplasmodia (-•›-) and swarm cells (- •œ•-). 258 T. Kuroiwa et al.

2b) and transmission phase contrast-epi-fluorescent micrographs (Fig. 2c) illustrating the cell nucleus, mitochondria and mt-nuclei in a plasmodium fixed 2 h after MII and stained with ethidium bromide. The phase contrast image gives a vague outline of the mitochondria but not of the mt-nuclei (Fig. 2a), while fluorescent microscopy does not show the outlines but does illustrate the rod-shaped mt-nuclei which have emitted red fluorescence (Fig. 2b). Phase contrast fluorescent microscopy proved to be the most useful technique of the three when observing mt-nuclei. Fig. 2c illustrates

Fig. 2. Phase contrast (a), fluorescent (b) and phase contrast fluorescent micrographs (c—i) illustrating mitochondria and their nuclei during S- (a—c, e, f), M- (g, h) and Gi-phases (d, i): Fixed at 2h

(e), 1 h (f) before MII and 2 h (g, h), 3 h (i) after MII and stained with ethidium bromide. Note: a, b, and c were the same field. All magnification. •~ 5600 Mitochondrial Nuclei in Live Cells 259 simultaneously the mitochondria and mt-nuclei in the same field as Fig. 2a and 2b. Ethidium bromide fluorescence was restricted to the cell nucleus and the mt-nuclei

(Fig. 2c). The cell nucleus appeared as a large spherical body. In general, the nucleoli, which occupy a central area of the nucleus, were not stained by the ethidium bromide although in the Fig. 2c fluorescence did not illustrate them either due to the presence of chromatin in the karyoplasm. On the other hand, the mt-nuclei appeared in the oval mitochondria as small rod-like structures emitting red fluorescence (Fig. 2c). The dividing mitochondria preserved their dumbbell structure in the buffer-S1. Figs. 2d to 2i are a series of phase contrast fluorescent micrographs of mitochondria containing mt-nuclei during mitochondrial G1 (Figs. 2d, i), mitochondrial S (Figs. 2e, f) and mitochondrial M phases (Figs. 2g, h), respectively (13). The small oval mitochondria become elliptical (Fig. 2e). The mt-nucleus elongates longitudinally during mitochondrial growth (Figs. 2f, g). When the mitochondrion had elongated and its major axis had reached approximately 3.5 ƒÊm, it took on a dumbbell-shape (Fig. 2h). Then it divided into two daughter mitochondria with separate mt-nuclei. The results obtained from the squashed preparations suggest that events leading up to mitochondrial division progressed normally. Based on results obtained from fixed cells, three stages mitochondrial S, mitochondrial M and mitochondrial G1 were selected as suitable for observations of moving mitochondria and their nuclei in live cells.

In a live plasmodium, only the mt-nuclei and not the cell nucleus could easily be stained with ethidium bromide (Fig. 3). The specific interaction between ethidium bromide and the mt-nuclei is as yet unclarified. Due to the rapid movements of mitochondria in the live plasmodia endoplasm caused by cytoplasmic streaming, neither the mitochondria nor their nuclei could be followed within one visual field for longer than 20 sec. On the other hand, it took 10-15 min for mitochondria in the ectoplasm to be carried away by cytoplasmic streaming after the ectoplasm had solated. Accord- ingly, the mitochondria within the ectoplasm were observed. Mitochondria were stained with ethidium bromide concentrations of 5, 10, 50 and 100 ƒÊg/ml but the intensity at either 50 or 100 ƒÊg,/ml did not appear significantly greater than that at 10 ƒÊg/ml. Although some adverse effects were observed at the higher concentrations (50 and 100 ƒÊg/ml), there was no apparent toxicity in cells treated with 10 ƒÊg/ml for 1 h. Cells which were returned to the standard culture medium grew normally, as did the untreated control group.

Changes in mitochondrial distribution and orientation and changes in the shape of mt-nucleus were not recorded by phase contrast microcinematography because the mt-nucleus was small and the fluorescence was weak. Therefore, the behavior of mt-nuclei in live cells was recorded only by the night vision technique. Figs. 3a—e show a sequence of videotape frames taken of moving mitochondria and their mt-nuclei during the mitochondrial S phase in a plasmodium harvested 2 h before MII and stained by means of the double staining technique. It was difficult to visualize mitochondria in a thick microplasmodium by phase contrast microscopy (Fig. 3a). However, when the plasmodium in the same field was excited with blue light (450-nm), several spherical or ovoidal mitochondria, each about 1.3 p.m in diameter, and emitting the yellow fluorescence of DASPMI, appeared (Fig. 3b). The focus was on the mitochondria in the cytoplasmic area beneath the cell membrane and thus mitochondria in the same field, and not those in the focal plane, appeared smaller than natural. When the blue filter was replaced by a green one, the mitochondria disap- peared, and a rod-shaped mt-nucleus emitting red ethidium bromide fluorescence 260 T. Kuroiwa et al. Mitochondrial Nuclei in Live Cells 261 became visible inside each mitochondrion. The mt-nucleus was about 1 ƒÊm long and 0.3 ƒÊm wide, and thus similar in size and shape to the mt-nucleus during the S phase in fixed plasmodium (Fig. 2e). The change of orientation and position of mitochondria was seen under time lapse photography (Figs. 3c, d and e). Dumbbell-shaped mitochondria, in the process of dividing, appeared in plasmodium harvested 3 h after MII

(Figs. 3f-j). They moved around in the plasmodium and when a mitochondrion's long axis was perpendicular to the surface of the frame, it looked like a small spherical body (Fig. 3f). When the blue filter was replaced by a green one, a dumbbell-shaped mt-nucleus appeared within each mitochondrion (Figs. 3h, i and j). The mt-nuclei appeared to elongate in a longitudinal direction, to a point 1.5 times the length of the mt-nucleus in an oval mitochondrion. Unfortunately, the mitochondria flowed away before the termination of division when the ectoplasm in the field solated. An hour later the dumbbell-shaped mitochondria had decreased in number, while many small, spherical mitochondria had begun to appear in the plasmodium (Figs. 3k and 1). All of the later contained one rod-shaped mt-nucleus 0.7 ƒÊm long and 0.3 ƒÊm wide. Since these spherical mitochondria were one-half the volume of the dumbbell-shaped mitochondria, the latter would seem to have divided during this time. In addition, the homogeneity in volume of the mitochondria in one visual field suggests that growth and division occurred semi-synchronously. In the live cells, each small spherical mitochondrion elongates and grows into a dumbbell-shaped mitochondrion, and which then divides semi-synchronously into two spherical daughter mitochondria. The dumbbell-shaped nucleus also divides at this time into two equal parts and is appor- tioned into the daughter mitochondria.

DISCUSSION

Bereiter-Hahn (1) used DASPMI at concentrations of 1.9 pcM as a fluorescent probe for in situ mitochondria in cultured cells. In this experiment, the Physarum mitochondria did not stain at such low levels. When the mitochondria were stained with 50 ƒÊg/ml DASPMI, higher fluorescent intensity was obtained although the dif- ference between this and that of Bereiter-Hahn is unknown. In a previous study (14), when Physarum mitochondria were isolated from the plasmodia using a 0.25 M sucrose buffer, the dumbbell-shaped mitochondria swelled into large spherical bodies and the dividing mt-nuclei were deformed and V-shaped. In the present study the mitochondria preserved their shape when a buffer-S1, containing 0.75 M sucrose was used. This buffer seems to be more suitable if mitochondria are to remain intact. Kuroiwa et al. (13) using thin sectioning techniques, demonstrated that small oval mitochondria became elliptical and the mt-nucleus elongated in a longitudinal direction during mitochondria' growth. When the mitochondrion elongated and its major axis reached approximately 3.5 ƒÊm, the mitochondrion took on a dumbbell-shape. Then it divided into two daughter mitochondria with separate mt-nuclei. The results obtained here by double fluorescent staining agree with the previous observations. It is possible to apply this method to Allium cepa, a higher plant, and to Euglena

Fig. 3. Three series of multiple video frames of ectoplasm regions in Physarum polycephalum harvested from the same live plasmodium 2 h before M‡U (a-e), 3 h after M‡U (f-j)and 4 h after M‡U (k-o). All were stained with ethidium bromide and DASPMI. Successive photographs were taken at 30-sec intervals. x 3800 262 T. Kuroiwa et al. graciris, Tetrahymena pyriformis, unicellular protozoa, which contain a smaller amount of DNA (unpublished data) than does Physarum polycephalum. The double staining technique appears to be a powerful tool to understand the biology and physiology of mitochondria] migration and the mitochondrial division cycle. Acknowledgements.This work was supportedin part by Grants-in-Aidfor Scientific Research Nos. 521708and 511212from the Ministry of Education, Scienceand Culture, Japan.

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(Received for publication, July 1, 1981)