fluorescence microscopic studie of s mitochondrial ... · tion of 500 iig/m\. the fluorescent...

J. Cell. Sci. 66, 21-38 (1984) 21Printed in Great Britain © The Company of Biologists Limited 1984

FLUORESCENCE MICROSCOPIC STUDIES OF

MITOCHONDRIAL NUCLEOIDS DURING MEIOSIS

AND SPORULATION IN THE YEAST,

SACCHAROMYCES CEREVISIAE

ISAMU MIYAKAWA, HIROYUKI AOI, NOBUNDO SANDOBiological Institute, Faculty of Science, Yaniaguchi University, Yamagucht 753, Japan

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

SUMMARY

Configurational changes of mitochondria and mitochondrial nucleoids (mt-nucleoids) duringmeiosis and sporulation in the yeast, Saccharomyces cerevisiae, were examined using the mito-chondrial membrane-binding fluorescent dye, dimethyl aminostyrylmethylpyridiniumiodine(DASPMI) and the DNA-binding fluorescent dye, 4',6-diamidino-2-phenylindole (DAPI).

In zygotes just after mating, mt-nucleoids were observed as many small discrete light spots in thecytoplasm. During meiosis in zygotes, mt-nucleoids at first coalesced with each other into a longstring and then separated into spherical nucleoids in four spores. These changes paralleled those inmitochondria observed using DASPMI.

The use of spheroplasts allowed us to examine the behaviour of mt-nucleoids at higher resolutionand to identify several distinct meiotic prophase stages of the cell nucleus during early sporulation.In diploid spheroplasts at the stationary phase, 50-70 of the mt-nucleoids were observed to beseparated from each other and each spherical mitochondrion contained only one mt-nucleoid. At thelater stage of premeiotic DNA synthesis, a single branched giant mitochondrion was formed as aresult of complete mitochondrial fusion. All of the mt-nucleoids were arranged in an array on a giantmitochondrion and coalesced into a string-like network. Through meiosis I and II, strings of mt-nucleoids were observed close to the dividing nuclei. At late meiosis II, a ring of mt-nucleoidsenclosing each daughter nucleus was formed. In ascospores, discrete small nucleoids were visibleclose to each spore nucleus with a 'string-of-beads' appearance. Many mt-nucleoids were excludedfrom the ascospores and remained in the residual cytoplasm of the ascus.

INTRODUCTION

Considerable knowledge of mitochondrial genetics in the yeast, Saccharomycescerevisiae, has been accumulated recently (see reviews by Birky, Acton, Dietrich &Carver, 1981; Dujon, Slonimski & Weill, 1974; Gillham, 1974; Dujon, 1981). Fromgenetic studies with drug-resistant mitochondrial markers recombination betweenmitochondrial genes has been demonstrated (Thomas & Wilkie, 1968; Coen et al.1970). These features have been incorporated into a general model for recombinationand segregation of mitochondrial genes (Dujon et al. 1974). The model proposes apanmictic pool of DNA molecules in which pairing and recombination events takeplace. To date, no structural observations have been made that provide an under-standing of the mechanisms of the model.

Studies of mitochondrial biogenesis have been based on the examination of serial

22 /. Miyakawa, H. Aoi, N. Sando and T. Kuroiwa

ultrathin sections by electron microscopy and analysis of three-dimensional recon-stitution. From these studies it has been shown that the number, form and volume ofmitochondria are closely related to the life cycle and physiological state of the cell(Hoffman & Avers, 1973; Stevens, 1977, 1981).

On the other hand, recently, a DNA binding fluorochrome, 4',6-diamidino-2-phenylindole (DAPI) has been used for visualization of mitochondrial DNA at thelight microscopic level (Williamson & Fennell, 1975). In yeast cells, using the DAPIstaining technique, mitochondrial nucleoids (mt-nucleoids) have been seen as smalldiscrete fluorescent spots and termed 'chondriolites' (Williamson, 1976). Chon-driolites have been observed with a string-of-beads appearance during exponentialgrowth (Williamson & Fennell, 1975). During meiosis-sporulation of the whole cells,as we reported previously, mt-nucleoids also aggregate and form structures with astring-of-beads appearance and then separate again into individual chondriolites ineach spore (Sando, Miyakawa, Nishibayashi & Kuroiwa, 1981). The mechanism ofdistribution of mt-nucleoids during meiosis in diploid cells, especially in the zygote,poses an important question in relation to genetic studies. However, the rigid cell wallprevented us from observing the fine structure of mt-nucleoids and an improvedmethod was needed. We used synchronous sporulation culture of spheroplasts for thepresent method, which enables us to examine mt-nucleoids at higher resolution. Inthis study, we demonstrate configurational changes of mt-nucleoids during meiosisand sporulation corresponding to the stages of meiotic nuclear division.

MATERIALS AND METHODS

Strains, media and growth conditionsSaccharomyces cerevisiae haploid strain 24428 (mating type a) and 3626 (mating type a) , and

a diploid strain G2-2 (a hybrid of 24428 with 3626) were used.For growth of haploid cells YEPD medium, containing 1 % yeast extract (supplied by courtesy

of Oriental Yeast Co., Ltd), 2% peptone and 2% glucose, was used. For the mating experiment,midlog-phase cells were harvested and cells of opposite mating types were mixed in equivalentamounts of approx. 1-5 X 107 cells/ml. Mating was carried out at 30°C in an enriched mediumcontaining 1% yeast extract, 2% peptone and 10% glucose, according to the method of Lee,Lusena & Johnson (1975). For sporulation, young zygotes were harvested 3h after mixing andinoculated into the same volume of sporulation medium containing 60mM-potassium acetate and50mM-potassium phosphate buffer (pH6'89). Sporulation culture of young zygotes was done at30 °C with reciprocal shaking.

Presporulation culture and sporulation culture methods for diploid whole cells were the same asthose used by Sando et al. (1981). Presporulation culture was performed at 30°C under reciprocalshaking for about 30 h. Cells at stationary phase were harvested and large cells of age one or abovewith bud scar(s) were separated by repeated differential and density gradient centrifugations, usingdextran (lot no. YH-5670, supplied by courtesy of Meito Sangyo Co., Ltd).

Preparation and sporulation culture of spheroplastsPreparation of spheroplasts was carried out by the method of Ueki, Shida & Tanji (1981), with

minor modifications. The large cells, at a concentration of 3 X 107 cells/ml, were incubated at30°C for 15min in a pretreatment solution containing 0'2M-2-mercaptoethanol, 0 8 M - K C 1 and25 mM-potassium phosphate buffer (pH75) . After washing with 25 mM-potassium phosphatebuffer (pH7-5) containing 0 -8M-KC1 by centrifugation at 700 |f for 5min, the cells (3 X 107

cells/ml) were treated at 30°C for 15 min with 200/ig/ml Zymolyase 60000 (Kirin Brewery Co.,

Mitochondrial nucleoids of yeast 23

Ltd) in 25 mM-potassium phosphate buffer (pH7S) containing 0 # 8 M - K C 1 . Spheroplasts werewashed with 25 mM-potassium phosphate buffer (pH 6-89) containing 0-8 M-KCI and immediatelyinoculated into sporulation medium containing 15 mM-potassium acetate, 50 mM-potassiumphosphate buffer (pH6'89), and 0'6M-sorbitol as an osmotic stabilizer. Sporulation culture ofspheroplasts was carried out at 30 °C with reciprocal shaking in 10 ml of sporulation medium in100 ml Erlenmeyer flasks at a cell density of 3 X 107 cells/ml.

Techniques of stainingSamples from the culture were taken at regular intervals after inoculation. For DAPI staining,

whole cells or spheroplasts were fixed by adding 1 % glutaraldehyde to the cultures at room tem-perature for lOmin. Specimens were washed once with NS buffer (20mM-TrisHCl (pH7-6),0'25 M-sucrose, 1 niM-EDTA, lmM-MgC^, O-lmM-ZnSCH, O-lmM-CaCU, 0'8mM-phenyl-methylsulphonylfluoride, 0 0 5 % 2-mercaptoethanol) (Sando et al. 1981) and suspended in thesame buffer. A drop of suspension containing fixed whole cells or spheroplasts was put on a glassslide, then a drop of DAPI (1 j/g/ml) dissolved in NS buffer was added, and they were thoroughlymixed. Then a coverglass was placed on the mixture and samples were squashed lightly on a glassslide.

For observation of the mitochondria in living cells, dimethyl aminostyrylmethylpyridiniumiodine(DASPMI), (first used by Bereiter-Hahn, 1976), was used. A drop of culture suspension containingwhole cells or spheroplasts was mixed with a drop of staining solution containing 500jig/ml ofDASPMI dissolved in NS buffer. Then a coverglass was placed on the mixture.

Fluorescence microscopyAll observations were made with an Olympus BHS-RFK epifluorescence microscope equipped

with a high-pressure mercury vapour lamp (HBO: 100W), an UVFL 100/130 objective lens,phase-contrast objectives and also an improved lens system to obtain stronger fluorescence. Ob-servations of fluorescence were made with a UVFL 100 objective using a blue excitation filter(420 nm) in combination with a 530 nm suppression filter for DASPMI and an ultraviolet excitationfilter (334nm) in combination with a 420nm suppression filter for DAPI. A combination of bothepifluorescence and transmission phase-contrast microscopy was used to detect the outlines ofmitochondria and the mt-nucleoids simultaneously. Photographs were taken at a magnification ofX850 on 35 mm Fuji Neopan (ASA 400) film with exposure times of 20s.

Measurement of nuclear DNA contentFluorescence microscopic photometry was used to measure nuclear DNA content according to

the method of Suzuki et al. (1982) with a minor modification. The spheroplasts were stained withpropidium iodide (10 fig/ml) dissolved in modified NS buffer, using 0-6 M-sucrose instead of0'25 M-sucrose, containing RNase A (Sigma Chemical Co., from bovine pancreas) at a concentra-tion of 500 iig/m\. The fluorescent intensity was measured in a Zeiss MPM 01K-FL/03FLepifluorescent microscope-photometer.

RESULTS

Meiotic behaviour of mt-nucleoids in zygote

A dumb-bell-shaped yeast zygote was formed by the fusion of two parental cells ofopposite mating types. The zygotes were transferred to sporulation medium soon aftermating, which occurred after 3 h of incubation in the mating medium. Fig. 1 showschanges in mitochondrial nucleoids during meiosis and sporulation of zygotes. Fig.1A, C, E, G are phase-contrast photomicrographs and Fig. 1B, D, F, H are DAPIfluorescence photomicrographs taken of the same fields. DAPI fluorescence wasrestricted to the cell nuclei and mt-nucleoids, which were sensitive to DNase (data not

/. Miyakawa, H. Aoi, N. Sando and T. Kuroiwa

•*? m


shown). During mating, nuclear fusion occurred immediately after cell fusion in thecentral region of zygote. Although the mt-nucleoids had a strings-of-beads appearanceduring vegetative growth of haploid cells, the strings of beads were dispersed intosmall spherical particles as soon as zygotes were formed. As seen in Fig. 1B, in a zygotejust after mating the cell nucleus appeared as a large spherical body and the mt-nucleoids were observed as small light spots separate from each other in thecytoplasm. In whole cells, however, it was difficult to identify the various stages, suchas meiotic prophase and metaphase. After inoculation into the sporulation medium,mt-nucleoids began to fuse with each other and a fluorescent network of mt-nucleoidswas formed in 7 h cultured zygotes (Fig. ID). At the first nuclear division, about 12 hafter inoculation, large long mt-nucleoids were seen closely surrounding two daughternuclei. At the second nuclear division, about 15 h after inoculation, mt-nucleoidsproduced a striking X-shaped configuration in the central region between four nuclei.Each end of the X-shaped configuration extended towards each of the four nuclei (Fig.1E, F). At 24h after inoculation, in the ascospore, mt-nucleoids closely encircled thecell nucleus in a strings-of-beads appearance (Fig. 1G, H). Many mt-nucleoids wereexcluded from the ascospores and remained in the residual cytoplasm of the ascus(arrowheads in Fig. 1 H) . These observations on zygotes agreed well with the previousreports (Sandoe/ al. 1981; Stevens, 1981) that described the changes of mt-nucleoidsduring sporulation of whole cells.

Fig. 2 shows DASPMI-stained mitochondria in zygotes at the same stages as thoseshown in Fig. 1A— H. Many small spherical mitochondria were dispersed in the zygotejust after cell fusion (Fig. 2A, B). Then small spherical mitochondria fused with eachother to form long mitochondria with branched structures. The filamentous or mesh-like structure of fused mitochondria extended from one end of the zygote to the otherduring meiosis (Fig. 2C-F) . In ascospores, small spherical mitochondria were locatedin an array like strings of beads at the peripheral region (Fig, 2G, H). The presentresults show that fusion and division of mt-nucleoids observed by DAPI stainingparalleled these morphological changes of the mitochondria.

Meiotic behaviour of mt-nucleoids in diploid spheroplasts

In order to identify the nuclear stages and to confirm the behaviour of mt-nucleoidsduring the meiotic process at higher resolution, sporulation and staining with DAPIwere carried out in spheroplasts from diploid cells. A diploid strain G2-2 was obtainedby crossing strain 24428 with strain 3626 of the opposite mating type. Vegatativediploid cells at stationary phase were fractionated and the large cells were convertedto spheroplasts.

Fixed and squashed spheroplasts enabled us to visualize mt-nucleoids and the cellnucleus at higher resolution than that for whole cells.

Fig. 1. Phase-contrast (A, C, E, C) and fluorescence (B, D, F, H) photomicrographs of thesame fields showing cell nuclei (/;) and mt-nucleoids (arrowheads) in zygotes duringmeiosis and sporulation. Zygotes were fixed at 0 (A, B), 7 (c, D), IS (E, F) and 24h (c, H)after inoculation to the sporulation medium and stained with DAPI. These zygotes wereobserved as whole cells. X3740.

2 CEL66


Fig. 2


Fig. 3 shows serial configurational changes in mt-nucleoids and the cell nucleusduring meiosis and sporulation of spheroplasts. At stationary phase (Fig. 3A), thechromatin in the spherical nucleus was observed to be amorphous and small sphericalmt-nucleoids were observed separate from each other in the cytoplasm. Thedistribution of mt-nucleoids might not be random but they may be partially aligned.The number of mt-nucleoids was 50—70 per protoplast and the diameter was about0'3jLim. After 2h of sporulation culture, neighbouring mt-nucleoids began to alignand fragments of strings of beads were formed (Fig. 3B). At 4h after inoculation, inmany cells the chromatin in the nucleus remained amorphous but the nucleus wascharacterized by expansion and seemed to be in premeiotic S-phase as seen bymicroscopic photometry, as described below (Fig. 4). All of the mt-nucleoids in aprotoplast were organized in an array and looked like a string of long mt-nucleoids(Fig. 3c). A string of mt-nucleoids showed some branching to form a fluorescentnetwork (Fig. 3c). After 6 h of sporulation culture, in the cell nucleus, as a result ofchromatin condensation a chromonema-like fibre of about 0-3 Jim in diameter, whichis about the same thickness as a string of mt-nucleoids, became visible (Fig. 3D). DNAin long mt-nucleoids stained homogeneously rather than as a strings of beads, suggest-ing that small mt-nucleoids had fused linearly (Fig. 3D). At 8h, in most of thespheroplasts, condensation of chromatin fibres increased and a chromosome-likestructure appeared in the nucleus (Fig. 3E). AS the meiotic stage proceeded,chromosome-like structures were observed separate from each other (Fig. 3F).Although mt-nucleoids remained as a branched string, a network of mt-nucleoids thathad become localized in part of the cytoplasm was frequently observed (Fig. 3E, F),and in some spheroplasts partial fragmentation of a network was observed (Fig. 3G).At the time of the first nuclear division, mt-nucleoids were observed close to thenucleus. The branches of a network closely encircled the dividing nucleus (Fig. 3H).At the time of the second nuclear division, which followed closely after the firstnuclear division, the string as a whole showed increased fluorescence and formeduniform wide bands. The bands of mt-nucleoids produced a striking X-shaped con-figuration in the central region between four nuclei. Each end of the X-shaped con-figuration of mt-nucleoids was extended towards each of the four nuclei (Fig. 3i). Inspheroplasts that had completed the second nuclear division, a ring of mt-nucleoidsenclosed each daughter nucleus (Fig. 3j). Inasci, a ring of mt-nucleoids incorporatedin each spore divided into small spherical mt-nucleoids and were visible close to each

Fig. 2. Phase-contrast (A, C, E, G) and fluorescence (B, D, F, H) photomicrographs of thesame fields showing mitochondria (arrowheads) in zygotes during meiosis and sporulation.Zygotic whole cells were vitally stained with DASPMI at 0 (A, B), 7 (c, D), 15 (E, F) and24 h (G, H) after inoculation to the sporulation medium. X3740.

Fig. 3. Serial fluorescence photomicrographs showing cell nuclei and mt-nucleoids inspheroplasts during meiosis and sporulation after DAPI staining. Spheroplasts at station-ary phase (A) and spheroplasts during sporulation were fixed at 2 (B), 4 (c), 6 (D), 8 (E,F, c), 10 (H), 12 (i, j) and 24 h (K, L), respectively, after inoculation into the sporulationmedium. The phase-contrast photomicrograph (L) shows the same field as (K). A-C, G-L,X3740; D - F , X4680.


Fig. 3A-F . For legend see p. 27.


Fig. 3 C - L . For legend see p. 27.

30 /. Miyakawa, H. Aoi, N. Sancto and T. Kuroiica

1001 z—" 150

8 12 16Culture time (h)

20 24

Fig. 4. Development of the meiotic process in spheroplasts from large diploid cells.Relative fluorescence intensity of nucleus measured by fluorescence microscopicphotometry (O O), percentage of mature asci (A A) and of cells that passed earlyprophase ( • • ) , late prophase or metaphase I (D—•—•), first nuclear division( • • ) , and second nuclear division (A A), are presented. The timing of meioticstages shown in Fig. 6 was defined as the time at which approximately 50 % of thespheroplasts have attained each level of development.

spore nucleus in a string-of-beads appearance (Fig. 3K). From fluorescent and phase-contrast microphotographs of the same fields, it was confirmed that many mt-nucleoids were excluded from the ascospore and remained in the residual cytoplasmof the ascus (Fig. 3K, L). The configurational changes of mt-nucleoids revealed byDAPI staining were also observed by vital staining using the alternative fluorochromeethidium bromide (data not shown).

The kinetic changes in the sporulation process in the spheroplasts from large cellsare shown in Fig. 4. The sequence of the meiotic stages is basically similar in timingto that of whole cells. Fixed and squashed spheroplasts enabled us to visualize mt-nucleoids and the cell nucleus, especially at various stages such as meiotic prophaseand metaphase (Fig. 3D-G), at higher resolution than for whole cells. The cellnuclear DNA content measured by fluorescence microscopic photometry usingpropidium iodide doubled between 2 and 4h of sporulation culture, and this stagewas comparable to the stage of expansion of the cell nucleus observed by DAPIstaining. As various stage of meiotic prophase, such as leptotene, zygotene,pachytene, diplotene and diakinesis, have not been generally recognized in yeast bylight microscopy, it was necessary to define the cytological landmarks of meioticprophase. In this paper we have defined a stage when 0-3 /im chromonema-like fibresappeared in the nucleus as'early prophase' (Fig. 3D), and a stage when a chromosome-like structure became obviously visible as 'late prophase or metaphase I' (Fig. 3E, F,G). The timing of the meiotic stages was defined as the time at which approximately


50% of the spheroplasts have reached each level of development; these configura-tional changes of the cell nucleus and mt-nucleoids during meiotic process aresummarized diagrammatically in Fig. 5. The timing of each stage is: premeiotic 5phase, 3-2; early prophase, 4-6; late prophase or metaphase I, 6-7; the first nucleardivision, 10-9; the second nuclear division, 11-6; and the appearance of mature asci,14-5h.

As shown by DAPI staining (Fig. 3A-C), as a result of fusion of mt-nucleoids astring of mt-nucleoids appeared at later premeiotic 5 phase. Actually, it was under-stood that the formation of long mt-nucleoids accompanied the formation of a giantmitochondrion, by following two methods of phase-contrast microscopy andDASPMI staining. In spheroplasts, the cell nucleus and mitochondria were easilysquashed out of the cells. Fig. 6A, B are phase-contrast and fluorescence photomicro-graphs of a squashed spheroplast at stationary phase. In Fig. 6B, a large brightspherical body is the cell nucleus and the small spots are mt-nucleoids. In phase-contrast microphotographs, spherical and less dark particles can be distinguishedfrom darker ones; the spherical particles are mitochondria and the darker particles areprobably lipid granules. The small spherical mitochondria were dispersed in thespheroplasts and each mitochondrion contained only one mt-nucleoid (Fig. 6A, B). At4h after inoculation, a single giant mitochondrion was formed as a result of thecomplete fusion of all the mitochondria. The length of the giant mitochondrion wasabout 60 nm (Fig. 6c). Mt-nucleoids appeared on the giant mitochondrion in an arrayand fusion of neighbouring mt-nucleoids seemed to have occurred (Fig. 6D). It couldbe seen that in a part of the giant mitochondrion, long mt-nucleoids were broken orhad not fused with each other (arrows in Fig. 6D). Occasionally faintly yellowishfluorescent spots were observed beside the string of bright blue-white fluorescence.As these coincided with the darker particles seen by phase-contrast microscopy, it maybe concluded that the yellowish spots were not mt-nucleoids (Fig. 6c, D).

Fig. 7 shows living spheroplasts stained with DASPMI. At stationary phase, smallmitochondria dispersed in spheroplasts were observed (Fig. 7A). On the other hand,at 4 h after inoculation, mitochondria fused to form long ones at the peripheral regionof the protoplast (Fig. 7B). These mitochondrial structures are consistent with thoseshown in Fig. 6A-D.

Fig. 5. Time-table of meiotic events in a synchronous culture of spheroplasts from largecells. Configurational changes of the cell nucleus and mt-nucleoids are illustrated diagram-matically. The nuclear stages depicted are, from left to right: stationary phase, premeioticS phase, early prophase, late prophase or metaphase I, first nuclear division, secondnuclear division and mature asci.

I. Miyakawa, H. Aoi, N. Sando and T. Kuroiwa

Fig. 6


Fig. 7. Fluorescence photomicrographs (A, B) showing mitochondria in spheroplasts atstationary phase (A), and after 4h of culture in sporulation medium (B), after DASPMIstaining. X3740.

DISCUSSION

Yeast DN A is mainly found in the cell nucleus and the mitochondria. For visualiza-tion of mitochondrial DNA (mtDNA), Williamson & Fennell (1975) first used aDNA-binding fluorescent dye, DAPI. This agent has a high affinity for AT-richDNA and produces a particularly strong fluorescence with yeast mtDNA, in situ aswell as in the cell-free state. By using DAPI, discrete fluorescent spots were distin-guished in the cytoplasm and termed 'chondriolites' (Williamson, 1976). Morpho-logical changes of chondriolites have been reported in vegetative cells (Williamson& Fennell, 1975) and in sporulating cells (Sando et al. 1981; Stevens, 1981). How-ever, previous studies did not satisfactorily determine the number and arrangementof mt-nucleoids accurately, because of the lack of resolution in microscopy of wholecells.

Accordingly, the prime consideration in our studies was to develop an improvedmethod that would facilitate the investigation of the structural properties of mt-nucleoids. For DAPI staining Kuroiwa & Suzuki (1980) first tried protoplasts of plantcells from Vi da fab a and Pisum sativum. In yeast cells, the use of spheroplastsallowed us to observe mt-nucleoids at higher resolution. Spheroplasts could be easilysquashed on a glass slide and the halation of fluorescence due to the cell wall disap-peared. The difference in resolution between whole cells and spheroplasts is obviousby comparing Fig. 1B, D, F, H with Fig. 3A-K. In addition, to avoid the physiological

Fig. 6. Phase-contrast (A, C) and fluorescence (B, D) photomicrographs of the same fieldsshowing cell nucleus (n), mitochondria and mt-nucleoids (arrows) at stationary phase (A,B), and a giant mitochondrion and mt-nucleoids (gaps between mt-nucleoids are shown byarrows in D) after 4h of culture in sporulation medium (c, D). These spheroplasts werespecially squashed and stained with DAPI. X3860.

34 /. Miyakawa, H. Aoi, N. Sando and T. Kuiviiva

changes during preparation of spheroplasts, synchronous sporulation culture ofspheroplasts instead of whole cells was carried out. By this method, not only con-figurational changes of mt-nucleoids but also the process of chromosome condensationand separation in the nucleus during meiosis could be observed.

The detailed meiotic process in the nucleus in S. cerevisiae has been elucidated onlyby electron microscopy on the behaviour of spindle pole bodies and the appearance offine structures like synaptonemal complexes (see reviews by Byers, 1981; Esposito &Klapholz, 1981). At the light microscopic level, using Giemsa staining, several stagesduring meiotic process have been defined (Tamaki, 1965; Simchen, Pinon & Salts,1972). Recently, Williamson et al. (1983) showed, using D API-fluorescent staining,five meiotic stages including premeiotic S, tentative pachytene, the first nucleardivision and the second nuclear division. They distinguished between premeiotic 5and tentative pachytene by the difference in the chromatin condensation. Althoughthe classical stages of meiotic prophase have been observed electron microscopicallyin S. cerevisiae] i.e. leptotene, zygotene, pachytene, diplotene and metaphase I(Zicker & Olson, 1975), no fine detail of chromatin condensation during meioticprophase and metaphase has been yet revealed at the light microscopic level. In thisstudy we could distinguish, as transient cytological landmarks, premeiotic S, earlyprophase and late prophase or metaphase I. The terms, 'early prophase' and Mateprophase or metaphase I' seem to be correct at present, but involve some ambiguity,because it is difficult to correlate precisely the stages observed in the light microscopewith various stages revealed by electron microscopy. However, from the similarity instructure to that of higher organisms and the comparison with the timing of the meioticprocess revealed by electron microscopy, it is assumed that early prophase probablyinvolves pachytene, and that late prophase or metaphase I involves diplotene,diakinesis and metaphase I, defined by the behaviour of spindle pole bodies and otherfine structures seen by electron microscopy.

In a recent study based on serial ultrathin sections, Stevens (1981) reported thatthere is one single highly branched mitochondrion per cell during meiosis and sporula-tion. According to this study, the chondriome is single throughout all the stages ofmeiosis and up to the formation of the four ascospores, and each ascospore alsocontains a single, branched mitochondrion that closely encircles the nucleus.

In our studies on mt-nucleoids, a complete fusion of mt-nucleoids occurred at thelater stage of premeiotic DNA synthesis, and throughout meiosis a network of fluores-cent strings was observed. These results obtained on the behaviour of mt-nucleoidsare basically consistent with the morphological changes of mitochondria reported byStevens (1981). In our studies, partial fragmentation of the network took placefrequently at late prophase or metaphase I. Just before spore wall formation, a strikingring structure of mt-nucleoids rather than a branched network was formed around thedaughter nucleus. In asci, fluorescent particles of mt-nucleoids were detected in afolded string-of-beads configuration in one or two circles around the nucleus.

Realistic present estimates of the number of 'chondriolites' suggest that there arearound 30 in a haploid cell (Williamson, 1976). In our strain, the number of mt-nucleoids in a diploid cell during stationary phase reached approximately 50-70 per


spheroplast from large cells. This value is close to that estimated by Brewer & Fang-man (1980) (50 copies of mtDN A per diploid cell). From these results, it is suggestedthat, in these culture conditions, most mt-nucleoids in stationary phase cells com-prise, on average, one or two mtDNA molecules.

On the other hand, our observations of the meiotic behaviour of mt-nucleoidsstrongly support the 'fluid' concept of the mitochondrial system (Williamson, 1976).From the genetic analysis (Wilkie, 1970), there is no doubt that recombination during

Haploid cell

'ulation I Zygoteium \ • ' " N

Speculationmedium

Completedsecond division

Ascus1 ate prophase or metaphase I

Fig. 8. A diagram of mt-nucleoid fusion and segregation during mating and meiosis-sporulation in zygote. Although fused mt-nucleoids were accumulated on a thread-likemitochondrion in exponentially growing haploid cells, dispersion of mt-nucleoids intomany small spherical particles (chondriolites) and a mixing of mt-nucleoids from bothparents occurred just after mating. During meiosis and sporulation in zygote, mt-nucleoids were arranged in an array on a fused mitochondrion, and recombination of DNAmolecules between neighbouring mt-nucleoids took place as a result of mt-nucleoid fusion.In mature ascosporcs mt-nucleoids again divide into chondriolites. tn, mitochondria; mn,mitochondrial nucleoids; n, cell nucleus; szv, spore wall.

36 /. Mivakawa, H. Aoi, N. Sando and T. Kumiwa

meiosis takes place between neighbouring mt-nucleoids in a fused mitochondrion.Although the timing of recombination of mtDN A cannot be discussed in this article,it is possible that the recombination of mtDNA occurs simultaneously at the time ofnuclear recombination before commitment to meiosis (Esposito & Esposito, 1974).Replication of mtDNA may occur in fusing mitochondria before premeiotic 5 phaseand also in a giant mitochondrion, since it is known from the biochemical analysis thatmtDNA synthesis starts before chromosome replication and continues at anapproximately constant rate throughout meiosis (Kiienzi & Roth, 1974).

This behaviour of mt-nucleoids poses a more important question for zygote forma-tion and sporulation. Hypothetical configurations of mt-nucleoids during zygoteformation and sporulation are illustrated in Fig. 8. In the zygote, the fine structure ofthe cell nucleus during meiotic prophase and metaphase I could not be clearly observedbecause of the rigid cell wall. However, our observations support the view that theprocess of fusion and partition of mt-nucleoids in the zygote might be basically thesame as that observed in sporulating spheroplasts from vegetative diploid cells. Themodel for mitochondrial recombination put forward by Dujon et al. (1974) centresround the idea that the mtDNA molecules in the zygote form a panmictic pool andundergo multiple random rounds of recombination. Although mtDNAs aggregated asfluorescent DNA, which accumulated on a thread-like mitochondrion in exponentiallygrowing cells (Sando et al. 1981), the dispersion of mt-nucleoids into many smallfluorescent ones just after mating may support the mixing of mitochondrial genes fromboth parents in the zygote. However, there is no cytological evidence as to how quicklyand how uniformly mt-nucleoids from both parents are mixed in the process of zygoteformation. If the mt-nucleoids are fully mixed in the zygote cytoplasm, a randomarrangement of mt-nucleoids from both parents on a giant mitochondrion would allowgreat freedom of access between mtDNAs and extensive recombination of neigh-bouring mtDNAs. On the other hand, incomplete mixing of mtDNAs may account foran effect of the bud position (central or apical) on the frequency of recombinants andparental classes (Strausberg & Perlman, 1978) and uniparental inheritance ofmitochondrial genomes during sporulation in young zygotes (Uchida & Suda, 1978).In the latter case, extrusion of many mt-nucleoids from the ascospores into theepiplasm may be related to some selection mechanism. Further cytological studies ofthe location and distribution of the mitochondrial DNA are needed to provide a com-plete explanation of the genetic observations.

We are indebted to Hiromu Abe, Akira Nomura, Natsuko Shohji and Kohtaroh Ohnuma forexploring the method of inducing synchronous sporulation in yeast spheroplasts.

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(Received 5 July 1983-Accepted 5 September 1983)

fluorescence microscopic studie of s mitochondrial ... · tion of 500 iig/m\. the fluorescent...

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