mitosis in the fission yeast schizosaccharomyces pombe as

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J. Cell Sd. 80, 253-268 (1986)
253
Printed in Great Britain © The Company of Biologists Limited 1986
MITOSIS IN THE FISSION YEAST
SCHIZOSACCHAROMYCES POMBE AS REVEALED BY
FREEZE-SUBSTITUTION ELECTRON MICROSCOPY
KENJI TANAKA* AND TOSHIO KANBE
Laboratory of Medical Mycology, Research Institute for Disease Mechanism and Control,
Nagoya University School of Medicine, Tsuruma 65, Shotua-ku, Nagoya 466, Japan
SUMMARY
Nuclear division in Schizosaccharomyces pombe has been studied in transmission electron
micrographs of sections of cells fixed by a method of freeze-substitution. We have found cytoplasmic microtubules in the vicinity of the spindle pole bodies and two kinds of microtubules, short
discontinuous ones and long, parallel ones in the intranuclear mitotic spindle. For most of the time
taken by nuclear division the spindle pole bodies face each other squarely across the nuclear space
but early in mitosis they briefly appear twisted out of alignment with each other, thereby imparting
a sigmoidal shape to the bundle of spindle microtubules extending between them. This configuration is interpreted as indicating active participation of the spindle in the initial elongation of the
dividing nucleus. It is proposed that mitosis is accompanied by the shortening of chromosomal
microtubules simultaneously with the elongation of the central pole-to-pole bundle of microtubules
of the intranuclear spindle. Daughter nuclei are separated by the sliding apart of interdigitating
microtubules of the spindle at telophase. Some of the latter bear dense knobs at their ends.
INTRODUCTION
Mitosis in Schizosaccharomyces pombe has been studied with light and electron
microscopy by McCully & Robinow (1971). Mitotic chromosomes were neither
identified in the nuclei of living cells nor in electron micrographs, but light microscopy of stained preparations showed them grouped in two clusters at opposite
poles of dividing nuclei. The authors believed the chromosomes to be directly
attached to the spindle pole bodies (SPBs), which, following Girbardt (1968), they
more specifically referred to as 'kinetochore equivalents' (KCEs). They attributed
division and separation, and thus the segregation of the chromosomes, chiefly to
expansion of the region of the nuclear envelope between the SPBs and opposite the
nucleolus. They assigned only a passive, scaffolding role to the intranuclear spindle,
which in most of their micrographs appears as a straight bundle of parallel microtubules (MTs). The behaviour of chromatin in S. pombe has been further studied by
Toda et al. (1981) with the help of the DNA-binding fluorescent probe DAPI. The
work of these authors confirms that the configuration of the chromatin compartment
of the nucleus is that ascribed to it, on the basis of electron micrographs, by McCully
& Robinow (1971).
• Author for correspondence.
Key words: mitosis, Schizosaccharomyces pombe, microtubules, freeze-substitution, electron
microscopy.
254
K Tanaka and T. Kanbe
A great advance in our understanding of mitosis in yeast Saccharomyces cerevisiae
is due to Peterson & Ris (1976). These authors demonstrated convincingly, with the
help of series of longitudinal sections and cross-sections, that in 5. cerevisiae the
central pole-to-pole bundle of MTs of the intranuclear spindle is flanked by short,
divergent MTs whose number in haploid cells as well as in diploids is in good
agreement with the known number of linkage groups (w = 17) in S. cerevisiae. This
finding suggested to the authors that, apart from the failure of the chromosomes
to condense, mitosis in Saccharomyces follows a conventional course, with the
chromosomes attached by MTs (i.e. not directly) to the spindle poles. The presence
of wisps of electron-dense material between and around the ends of the short,
divergent MTs further strengthened the authors' proposal.
Mitosis infissionyeasts is unlikely to differ fundamentally from mitosis in budding
yeasts. The example of the fruitful work of Peterson & Ris (1976) has encouraged us
to think that scrutiny of serial sections of S. pombe nuclei, something that had not
been carried out before, might increase the probability of finding the few chromosomal MTs to be expected in S. pombe, in which n = 3 (Kohli et al. 1977). To
improve further our chances of not missing important details we have used the
freeze-substitution method of fixation advocated by Howard & Aist (1979) for better
than usual preservation of microtubules. Our investigation is still in progress. We
have indeed found what we interpret as chromosomal MTs but so far only in
longitudinal sections; cross-sections of dividing nuclei remain to be studied. The
purpose of the present paper is threefold. To demonstrate the preservation of the
natural shape of 5. pombe nuclei attainable by freeze-substitution, to demonstrate the
presence of short, diverging, discontinuous, probably chromosomal MTs, and lastly
to report that we have several times encountered sigmoidally twisted spindles in
otherwise well-preserved nuclei. The latter observation suggests to us that the
spindle in S. pombe may play a more active role in mitosis than that ascribed to it by
McCully & Robinow (1971).
MATERIALS AND METHODS
Materials
Sckizosacchantmyces pombe strain h90 was used. The strain was maintained at room temperature on MY agar, which contained 0-3 % malt extract (Difco), 0-3 % yeast extract (Difco), 0-5 %
peptone (Difco), 1 % glucose and 2 % agar.
Phase-contrast microscopy of living cells
Cells grown in MY broth at 28 °C overnight were used as an inoculum for 'spreading drop' slide
cultures according to Robinow (1975). Living cells growing in a thin film of MY medium containing 21 % gelatin were viewed with phase-contrast using Zeiss Ultraphot II microscope.
Photographs were taken with X100 objective in conjunction with OPTOVAR in position 1-25X,
which provided an initial magnification of 400x on 35 mm film (Fuji NEOPAN 400).
Freeze-substitution electron microscopy
For freeze-substitution cells were inoculated into MY broth and cultured overnight at 28 °C.
A drop of the culture was spread on the surface of rectangular 5 mm X 7 mm pieces of cellulose
Mitosis in S. pombe
255
tubing (Cellophane tubing-seamless, Union Carbide Co., U.S.A.) placed onto the MY agar and
was incubated for about 6h at 28 °C.
Minor modifications apart, freeze-substitution was carried out as described by Howard & Aist
(1979). Pieces of cellulose tubing on which cells were growing were quickly taken off the agar and
immediately plunged into melting Freon 23 cooled with liquid nitrogen. The frozen sample was
transferred to the substitution fluid, anhydrous acetone containing 2 % OsC^ and 0-05% uranyl
acetate, maintained at —79°C with solid COz/acetone, and left for 48 h. Then the samples were
transferred to —20°C for 2h, to 4°C for 1—1 -5 h and finally to room temperature for 30min. They
were rinsed four times with anhydrous acetone. The pieces of cellophane with cells attached were
infiltrated with increasing concentrations of Epon-Araldite in anhydrous acetone and finally with
100 % Epon-Araldite. Resin-infiltrated cellophane pieces were sandwiched between Teflon-coated
glass, polymerized at 70°C for 48 h, and were checked under phase-contrast to determine which
cells were well-frozen. Well-frozen cells were trimmed, mounted on resin blocks and thin sections
were cut with a diamond knife. Serial sections collected on Formvar-coated single-slot grids were
stained with uranyl acetate and lead citrate. They were viewed in a JEOL 100 CX or HITACHI
H-800 electron microscope operated at lOOkV.
RESULTS
Observations on the nuclei of living cells with phase-contrast microscopy
The log-phase cells had many vacuoles scattered throughout the cytoplasm, which
sometimes made it difficult to follow the behaviour of the nucleus. However, in
MY—gelatin medium vacuoles disappeared in due course and the nucleus stood out in
low density against the dark background of the cytoplasm. Fig. 1 shows a set of timelapse phase-contrast micrographs illustrating phases of nuclear behaviour during
mitosis in two cells of 5. pombe. The beginning of mitosis was difficult to detect but a
slight decrease in the density of the nucleolus indicated that the cell was going into
division. A distinct feature of the mitotic nucleus was a change in nuclear morphology from spherical to more or less rectangular or ellipsoidal (Fig. 1: 6, 20),
followed by elongation into a gourd form (Fig. 1: 12, 25). The nucleolus was
stretched out in the interior of the elongated nucleus. After the dumbbell stage had
been reached, separation into daughter nuclei had taken place (Fig. 1: 14). The
divided nuclei were seen at the end of the cell for some time after separation (Fig. 1:
20, 32) and moved backward later at the time of septum formation (Fig. 1: 52).
We tried to determine the time course of mitosis in 5. pombe, but it was found to be
difficult to define the exact time from the time-lapse photomicrographs for each stage
of mitosis, in particular the time when mitosis began. However, our preliminary
studies on five sets of time-lapse observations indicated that about 5 min were
required for the ellipsoidal nucleus to divide and separate into the two daughter
nuclei and about another 10 min before septum formation began.
Electron microscopy of mitosis in S. pombe
Fig. 2 shows part of a section with one cell about to be bisected by a transverse
septum and the nuclei of its two neighbours at interphase of mitosis. The nuclei in
all three cells have circular profiles. They contain eccentrically placed nucleoli
(as familiarity with the nuclei of living S. pombe would lead one to expect) whose
substance contrasts more strongly with the chromatin portion of the nucleus than it
does in sections of conventionally fixed 5. pombe. Vacuoles that appear to be filled
256
K. Tanaka and T. Kanbe
Mitosis in S. pombe
257
with dense material in conventionally fixed cells have a spongy structure in S. pombe
prepared by freeze-substitution (cf. McCully & Robinow, 1971;figs1-6, 20 and 21).
Ribosomes also appear uncommonly well preserved.
An SPB is located in a zone bounded by the nuclear envelope and a mitochondrion
(Fig. 3), an association already noted by McCully & Robinow (1971). Beneath the
SPB across the nuclear envelope there is some amorphous dense material that seems
to contain very short MTs (arrowheads in Fig. 3). The SPB is also associated with a
few cytoplasmic MTs. It has a dumbbell shape, with a long axis of 350 nm and short
axis of HOnm (Figs 3, 4). At the start of mitosis the SPB divides, with spindle MTs
developing between sister SPBs. In the earliest stages when the two SPBs are only a
short distance apart the spindle is composed of short divergent and longer pole-topole MTs (Fig. 5). At this stage of mitosis one of the SPBs invariably occupies a
position in the nuclear envelope close to the nucleolar region (Figs 5,6). The nuclei
at this stage have either rounded or oval contours. The growing spindle gradually
comes to consist of a slightly curved bundle of long, parallel, probably continuous
MTs and short discontinuous MTs. One of these (arrowheads in Fig. 7B) is seen
ending in a circumscribed area of dense material differing slightly in texture from the
rest of the nuclear contents and perhaps representing part of a chromosome.
In the series illustrated by Figs 8 and 9 the nucleus becomes ellipsoidal, with
slightly pointed poles. In Fig. 8 the nucleolus has just begun to be stretched out
along the spindle and in Fig. 9 nucleolar material invests the spindle along most of its
length, recalling fig. 35 of McCully & Robinow (1971) and several sets of observations on dividing nuclei of living cells of other species of fission yeasts (e.g. see
Robinow, 1980). In the nucleus of Fig. 8 the flat inner surfaces of the SPBs are no
longer oriented parallel to each other and the spindle MTs that arise or terminate
perpendicular to these surfaces compensate with a double twist for the changed
alignment of the SPBs. We have collected eight sets of sections illustrating this
remarkable, previously unrecorded configuration, which will be dealt with further in
the Discussion. Three short cytoplasmic MTs are associated with SPBs in Fig. 8c.
The process of constriction begun in the nucleus of Fig. 9 has been completed in Figs
10 and 11, where daughter nuclei are still connected by a narrow corridor containing
a few MTs, three of which bear knobs of dense material at their ends (Fig. 11).
Freeze-substitution frequently does not preserve membranes well and did not do so
in this instance, but we know from sections of a nucleus at a corresponding stage of
constriction (figs 37, 39 of McCully & Robinow, 1971) that a continuous envelope
Fig. 1. Time-lapse phase-contrast micrographs of the two cells undergoing mitosis.
Numbers at the top of each Figure give minutes elapsed since the first picture of the series
was taken. X36O0. The interphase nucleus is seen in the lower cell until 12min. The
beginning of mitosis is seen at Omin for the upper cell and 14min for the lower cell. The
nucleus takes a more or less rectangular shape (6 min for the upper and 20 for the lower
cell), and elongates into the gourd form (12min for the upper cell and 25 for the lower
one). The intermediate is shown in elongated ellipsoidal shape (9 min for the upper cell).
The two daughter nuclei produced by division move towards the ends of the cell (20 min
for the upper cell and 32 for the lower one) and turn back to the centre of the daughter cell
when the septum is formed (52min for the upper and the lower cells).
258
K. Tanaka and T. Kanbe
surrounds daughter nuclei as well as the narrow channel that still connects them.
Arrowheads in Fig. 10 point to profiles of the incipient transverse septum. A slightly
later stage of cell and nuclear division is represented by the cell on the right in Fig. 2.
Our observations and conjectures are summarized schematically in Fig. 12.
So far we have examined only sections cut parallel to the plane of the dialysis
tubing on which the yeasts were grown. We have therefore not yet been able to study
Mitosis in S. pombe
259
the cross-sections of mitotic spindles, which would be required for reliable counts of
the type and number of MTs involved. Investigations along this line are now in
progress.
DISCUSSION
The smooth contours of the nuclei in our sections of cells preserved by freezesubstitution recall those of the nuclei in living cells of 5. pombe examined by phasecontrast microscopy (Fig. 1) and suggest that the wrinkled, indented countours of
the nuclei in the electron micrographs published by McCully & Robinow (1971) are
artefacts of the conventional fixation procedure followed by these authors. The
ribosomes also seem particularly well defined in our sections. This is not, however,
true of plasma membrane and nuclear envelope, which, on the whole, are rather
indistinct in our material.
Better preservation than that achieved by our predecessors, combined with a
sufficient number of serial sections, has enabled us to demonstrate cytoplasmic MTs
in close vicinity to the SPBs (Figs 3, 4, 8B,C, 9A,B) as well as short, discontinuous
intranuclear MTs diverging from the spindle poles (Figs 5B,D, 7B,C). Freezesubstitution thus endows the mitotic spindle of S. pombe, at least in the early stages
of nuclear division, with a close resemblance to the mitotic spindle of 5. cerevisiae as
described by Peterson & Ris (1976). We regard the short intranuclear MTs as
chromosomal ones and are confident that further work will show that designation of
the SPBs as kinetochore equivalents (McCully & Robinow, 1971) was inappropriate.
In the majority of published light and electron micrographs of dividing yeast
nuclei the mitotic spindles, beyond a certain point of development, appear remarkably straight. Exceptions from this rule are provided by fig. 29 of McCully &
Robinow (1971) and fig. 7 of Byers & Goetsch (1975), which show slightly curved
spindles. In dividing nuclei we have repeatedly encountered spindles more strikingly
curved and twisted than either of the earlier examples and this has led us to conclusions regarding the mechanism of mitosis in S. pombe that differ from those
arrived at by previous students of this species, as well as of Saccharomyces. In
5. cerevisiae the spindle tends initially to be inclined at a large angle to the main axis
Fig. 2. A section of three S. pombe cells preserved by freeze-substitution. The cytoplasm
is packed with dense ribosomes. Mitochondria are seen as variously shaped profiles of low
density lacking defined internal structure. The cytoplasm also contains many vacuoles
filled with a spongework of dense matter. Nearly half of the volume of the nuclei is
occupied by nucleolar material of labyrinthine organization with more or less transparent
channels traversing a dense granular matrix. The third cell from the left has completed
mitosis and is about to be divided by an ingrowing transverse septum (arrowheads). Bar,
5/im.
Fig. 3. A section of an interphase SPB on the outer membrane of the nuclear envelope.
A mitochondrion (m) is close by and between them there is a microtubule. An arrowhead
shows the presence of very short MTs associated with the dense material across the
nuclear envelope beneath the SPB. Bar, 0-5/an.
Fig. 4. Glancing section of an interphase SPB of dumbbell shape. It is associated with
several cytoplasmic microtubules. Bar, 0-5 /ttn.
260
K. Tanaka and T. Kanbe
Mitosis in S. pombe
261
of the elongating nucleus (Robinow & Marak, 1966; Byers & Goetsch, 1974, 1975;
Peterson & Ris, 1976; King, Hymans & Luba, 1982). As it gets longer the spindle
becomes increasingly attenuated by the loss of more and more MTs and ends up in
the maximally stretched nucleus as a very weak reed indeed (King et al. 1982). For
these reasons it is commonly agreed that in Saccharomyces the intranuclear spindle
neither initiates nor sustains the marked elongation of the dividing nucleus. To
account for the work to be done in dividing the nucleus Peterson & Ris (1976) had
recourse to the modus operandi invoked for nuclear division by McCully & Robinow
(1971), namely differential expansion of the nuclear envelope. Our own observations
on 5. pombe provide no support for the concept of membrane growth as the prime
motor of nuclear division. We instead suggest that in 5. pombe nuclear division proceeds in three steps. A fleeting initial phase, when the spindle is actively elongating
faster than the long axis of the dividing nucleus, is followed by one in which there is
synchrony between the two processes and, finally, by constriction of the nucleus.
The first of these postulated phases would account for our repeated finding of Sshaped spindles. The forces involved in the initial moving apart of the SPBs in the
periphery of the nucleus remain unknown in S. pombe and other yeasts but plausible
speculations based on in vivo experiments may now be made on the mechanics of the
later phases of mitosis. Studies of the effects of microlaser burns on the course of
mitosis in nuclei of Fusarium (Aist & Berns, 1981) have engendered the view that the
nucleus is pulled, not pushed, apart by forces acting on the SPBs via 'astral', i.e.
cytoplasmic bundles of MTs, and that the spindle has the task of regulating, by the
rate of its extension, the strength of this pull and thus the speed of genome
segregation. That such a mechanism of balanced pulling and yielding may also
be at work in mitosis in budding yeasts has to be considered after the detection
by Kilmartin & Adams (1984), using immunofluorescence microscopy, of broad,
often very long tracts of MTs extending from the SPBs into the cytoplasm of
Saccharomyces. We have found profiles of a few MTs on the cytoplasmic side of the
Fig. 5. A-D. Four consecutive sections of a nucleus at an early stage of spindle formation.
MTs radiate from SPBs arranged opposite each other. Several MTs are grouped together
to form the continuous pole-to-pole spindle. Bar, 0"5 fiin.
Fig. 6. Two SPBs are joined by a slightly curved bundle of MTs in an oval nucleus with
eccentrically placed nucleolus. The SPBs appear as dense plaques within the nuclear
envelope. One of them is closer to the nucleolus than the other one. At the left end of the
spindle a few short diverging MTs can still be discerned. Bar, 1-0 /an.
Fig. 7. Five serial sections of a nucleus at a stage of division similar to that shown
in Fig. 6. Arrowheads in B point to ends of discontinuous diverging MTs. The one on
the left seems to be in contact with granular matter that differs in texture from the rest of
the nuclear contents and may represent part of a chromosome. Bar, 0-5 /an.
Fig. 8. A-C. Serial sections of an ellipsoidal nucleus with S-shaped central spindle. The
nucleolus has a loose texture and partly surrounds the spindle. The SPBs are pressed
closely to the nuclear envelope and are associated with a few cytoplasmic ('astral') MTs
(arrowheads in B,c). The length of the spindle is 4-0 /an. Bar, 1-0/an.
Fig. 9. A-C. Serial sections of a gourd-shaped nucleus entering the phase of constriction.
MTs of the spindle, still visible in C, are slightly curved. The nucleolus is more stretched
out than the one in Fig. 8 and appears wrapped around the spindle. The spindle is 4-5 /an
long. Bar, 1-0/an.
262
K. Tanaka and T. Kanbe
Fig. 7. For legend see p. 261
Mitosis in S. pombe
Fig. 8, For legend see p. 261
K. Tanaka and T. Kanbe
264
'• *%',**'*
<:
fr~ j'-
Fig. 9. For legend see p. 261
Mitosis in S. pombe
Fig. 10. A dividing nucleus of dumbbell shape. AITOWS point to the SPBs at either end.
The nucleolar material has been divided between the incipient daughter nuclei, which are
still joined by a narroj? spindle channel (a tube, in reality; see the text). The length of the
spindle is 8-2/an. Arrowheads point to invaginations of the plasmalemma where a
transverse septum is beginning to grow inward. Bar, 1-0 ^m.
Fig. 11. Higher magnification of the telophase spindle channel of another cell. It contains
a few MTs some of which seem in touch with the borders of the channel and bear dense
knobs at their ends. Bar, 1-0 fan.
265
K. Tanaka and T. Kanbe
266
D
Fig. 12. Diagrammatic representation of a conjugated course of mitosis in S. potnbe.
A. Early stage of spindle formation. B. The stage at which chromosomal tubules have
become polarized in opposite direction. C. The S-shaped spindle with chromosomal MTs
much shortened. D. Elongation and constriction of the nucleus which now assumes
gourd-shape. E. Further elongation of the nucleus and its constriction into a dumbbell
shape. Incipient daughter nuclei are still connected by a slender spindle channel (tube).
Mitosis in S. pombe
267
SPBs but it remains to be seen whether the Aist-Berns mechanism holds true also for
fission yeasts. The picture of mitosis in 5. pombe that may be inferred from our
sections is, so far, compatible with the concept of an initially actively pushing but
later 'regulating', gradually yielding spindle. However, mitosis mechanics here as
elsewhere can be satisfactorily explored only by experiment.
Chromosomes cannot be identified reliably in our sections but if the ends of the
short, diverging spindle MTs are taken as indicators of the disposition of the
chromosomes then it will be seen that genome segregation in S. pombe occurs very
early in the course of nuclear division. In this respect 5. pombe behaves like
Saccharomyces. Separation of incipient daughter nuclei is achieved by the sliding
apart of interdigitating MTs of the telophase spindle. The nature of the dense knobs
at the ends of some of these MTs is unknown.
We are grateful to Dr C. F. Robinow at the University of Western Ontario and Dr I. B. Heath at
York University in Canada for their valuable discussions and for their help in the preparation of
this paper. This work was supported by a grant for scientific research (no. 58480018) from the
Ministry of Education, Science and Culture and by a research fund from the Toray Science
Foundation.
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{Received 10 July 1985 - Accepted 29 August 1985)
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