Download Genetic Block of Outer Plaque Morphogenesis at the Second Meiotic

Journal of General Microbiology (1982), 128, 1309-13 17. Printed in Great Britain
1309
Genetic Block of Outer Plaque Morphogenesis at the Second Meiotic
Division in an hfdl-l Mutant of Saccharomyces cerevisiae
By S U S U M U O K A M O T O * t A N D T E T S U O I I N O
Laboratory of Genetics, Department of Biology, Faculty of Science, University of Tokyo,
Hongo, Bunkyo-ku, Tokyo 113, Japan
(Received 8 June 1981; revised 22 September 1981)
An hfdl-1 mutant of Saccharomyces cerevisiae, SOS4, characterized by predominant
production of two-spored asci at 29 " C , undergoes normal meiotic nuclear divisions and
produces four haploid nuclei, but only two non-sister nuclei among them are incorporated
into mature ascospores. Spindle pole bodies and prospore wall membranes at the second
meiotic division at 29 OC were observed in this mutant by electron microscopy. The spindle
pole body at one pole of each spindle had a normal outer plaque which was larger than the
inner plaque. At the other pole, the outer plaque was entirely absent and a normal prospore
wall membrane was not detected. It was concluded that at 29 O C the hfdl-1 mutation blocks
the morphogenesis of outer plaques and prospore wall membranes at the two non-sister poles
in the second meiotic division, and consequently only non-sister nuclei in the resulting meiotic
cell are incorporated into ascospores.
INTRODUCTION
Meiosis and ascospore formation in Saccharomyces cerevisiae provides an excellent model
system for the investigation of developmental pathways (Hartwell, 1974; Esposito & Esposito,
1975). When diploid cells of S. cerevisiae heterozygous for mating-type locus are transferred
into nitrogen-poor medium containing acetate as a carbon source they complete meiosis and
ascospore formation to form four-spored asci. However, asci containing less than four spores
can be produced physiologically and genetically (Takahashi, 1962; Haber & Halvorson,
1972; Tsuboi & Yanagishima, 1974; Davidow et al., 1980; Grewal & Miller, 1972; Moens et
al., 1974; Srivastava et al., 1981). Analyses o f abnormal asci are useful for elucidating the
cause-effect sequences in sporulation (Moens et al., 1977; Davidow et al., 1980; Okamoto &
Iino, 1981).
A mutant, SOS4, which predominantly produces two-spored asci, was isolated from a
homothallic diploid, MT 13 of S. cerevisiae, and its mutant phenotype (Hfd; High frequency
of dyad production) was shown to be caused by a single nuclear mutation, hfdl-I (Okamoto
8z Iino, 1981). The hfdl-1 mutant was distinct from other mutants in that it was deficient in
the incorporation of two non-sister nuclei in a post-meiotic cell into mature spores (Okamoto
& Iino, 198 1).
Electron microscopic studies on the incorporation of nuclei into ascospores in wild-type
diploids of S. cerevisiae have revealed a series of changes in intracellular structures (Moens,
1971; Moens & Rapport, 1971; Zickler & Olson, 1975; Horesh et al., 1979). Before the
second meiotic division, the outer plaques of spindle pole bodies are modified and become
larger than mitotic and first meiotic ones. At the second meiotic division, two spindles form in
? Present address: Laboratory of Molecular Genetics, Institute of Medical Science, University of Tokyo, PO
Takanawa, Tokyo 108, Japan
0022-1287/82/oooO-9990 sO2.00 0 1982 SGM
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S . OKAMOTO A N D T . IINO
a nucleus and the nucleus buds to form a lobe at one side of each spindle pole. Then, each
lobe is included by a prospore wall membrane which originates at the cytoplasmic side of
the outer plaque. Eventually, each nuclear lobe 'pinches off' and the prospore wall membrane
closes to form an immature ascospore. These observations suggest that the outer plaque plays
an essential role in the formation of a prospore wall membrane (Moens, 1971; Moens &
Rapport, 1971; Zickler & Olson, 1975; Horesh et al., 1979), and indicate that two sister
nuclei, one of which is incorporated into an ascospore in the hfdl-l mutant at 29 OC, share
the same spindle at the second meiotic division.
This led us to expect that structural abnormalities might be detected in the mutant cell at,
or near, one of the two poles of a second meiotic spindle. Therefore, we examined in serial
thin sections the structures near the poles of the second meiotic spindles in the hfdl-I mutant
and its parental strain. We report here results which indicate that the outer plaque at one of
the two poles of the second meiotic spindle is entirely absent in the mutant.
METHODS
Media. The composition of the presporulation medium (YPA) and the liquid sporulation medium (SPOL) were
described previously (Fast, 1973; Okamoto & Iho, 1981).
Strains. For electron microscopy, an hfdl-I mutant, SOS4 (hfdl-Ilhfdl-I), and its parent, MT13
(HFDI + / H F D l +), were used. The former produces about 87 % two-spored asci and the latter produces about
72% four-spored asci when sporulation is induced in SPOL at 29 "C (Okamoto & Iino, 1981). The genetic
backgrounds of these two homothallic diploids differ only in a single nuclear gene, hfdl (Okamoto & Iino, 198 1).
Electron microscopy of meiotic cells. SOS4 or MT13 cells were inoculated into 100 ml YPA at a concentration
of 1-2 x lo5 cells ml-l. After 15 h incubation at 29 "C, the cells were washed with 100 ml distilled water and
resuspended in 100 ml SPOL in a 1 1 flask. The flask was aerated on a Taiyo incubator (model M- 100N) at 29 "C
and at 100-120 strokes min-'. At 7 h after transfer of the cells into SPOL, 33 ml of the SPOL culture was
removed and 1 ml 70% (w/v) glutaraldehyde (Ladd Res.) was added. Cells were immediately pelleted and fixed
with 5 m14 % glutaraldehyde at room temperature overnight. The cell wall was subsequently digested by glusulase
(Endo Laboratory Inc.) according to the method of Zickler & Olson (1975) except that 0.5 M-dithiothreitol was
used instead of 0.1 M-p-niercaptoethanol. The cells were post-fixed with 4 % (w/v) osmium tetroxide for 2 h,
contrasted in 0.5% (w/v) aqueous uranyl acetate solution for 2h, dehydrated in a graded ethanol series and
embedded in Spurr's resin (Spurr, 1969). Serial sections were cut on a Reichert OMU2 ultramicrotome with a
glass knife and picked up with Formvar-coated single-hole grids. Sections were stained in saturated uranyl acetate
solution for 2 h and then with lead citrate for 5 min (Reynolds, 1963). Samples were observed in a JEM lOOC
electron microscope operating at an accelerating voltage of 100 kV.
RESULTS
The structures observed around five pairs of spindle poles in five, second meiotic cells of
strain MT13 and seven pairs of spindle poles in six, second meiotic cells of mutant SOS4 are
summarized in Table 1.
In strain MT13, two stages during the second meiotic division were discriminated based on
the presence or absence of prospore wall membranes as previously reported (Moens &
Rapport, 1971). For convenience, we call these stage I and stage 11, respectively. Cells at
stage I had no prospore wall membranes, but these developed in cells at stage 11. All spindle
pole bodies detected in MT13 cells, irrespective of the stage, had inner plaques and large outer
plaques characteristic of the second meiotic division (Fig. 1) and normal prospore wall
membranes developed at both poles in stage I1 cells. These observations are consistent with
the results reported on other wild-type strains (Moens & Rapport, 197 1).
In mutant SOS4 cells, three types of pairs of spindle pole bodies were detected (Table 1). In
five of the seven pairs examined, the outer plaque was entirely absent at one of the poles,
while the other pole had a normal outer plaque (Fig. 2). In one of seven pairs, the outer plaque
was entirely absent at one of the poles, while the other pole had an outer plaque smaller than
the normal outer plaque. In the remaining pair, the normal outer plaque was detected at one
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Meiosis in S . cerevisiae
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Table 1. Structures observed in the spindle pole area during the second meiotic division
Structures were identified with serial sections by electron microscopy. The two pole areas of each
spindle were designated as X and Y. The structures observed were a prospore wall membrane (PSW),
outer plaque (OP) and inner plaque (IP). Cells at stage I had no prospore wall membranes, but these
developed in cells at stage 11. The structures corresponding to 2a and 2b were observed in the same cell.
Strain
MT13
(+/+)
Stage
Observed
cell no.
I
I
I1
1
2
3
I1
I1
SOS4
(hfdl-1lhfdl-I)
I
I
I
I1
I1
I1
I1
4
5
1
2a
2b
3
4
5
6
Y
X
\
I
PSW
-
-
+
+
+
-
+
+
+
+
OP
IP
IP
OP
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
k
+, Presence of normal structures; k,presence of smaller structures than those indicated as
structures; A, presence of abnormal structures.
*
-
PSW
-
+
+
+
-
A
-
A
-
A
-
A
+; -,
absence of
of the poles and the other pole had an outer plaque which was smaller and thinner than
normal. Four of the seven pairs described above corresponded to stage 11. In these pairs, an
abnormal prospore wall membrane, which appeared to be less dense and less rigid than the
normal prospore wall membrane, developed near the pole where the outer plaque was entirely
absent (Figs 3 and 4). In addition to these seven pairs of spindle poles, one spindle pole which
had a normal inner plaque, an outer plaque and a prospore wall membrane was detected in
both cell no. 3 and cell no. 4 of Table 1. One which had a much smaller and thinner outer
plaque than that detected in pole area X of cell no. 6 was detected in the same cell.
DISCUSSION
In each of the four pairs of spindle poles observed in the hfdl-I mutant cells at stage 11,
there was a normal prospore wall membrane at one pole but not at the other. In two of three
pairs observed at stage I, one spindle pole had a normal outer plaque and the other did not.
Only the former is expected to develop a normal prospore wall membrane at the subsequent
stage. These results are consistent with the previous conclusion that the two non-sister nuclei
were incorporated into ascospores (Okamoto & Iino, 1981). The exceptional one in which
outer plaques were detected at both poles at stage I, may correspond to a minor fraction
producing three-spored asci. Therefore, we conclude that the genetic block caused by the
hfdl-1 mutation at 29 OC affects the morphogenesis of outer plaques and prospore wall
membranes at the non-sister poles of the second meiotic spindle.
At a late phase of the first meiotic division, each spindle pole duplicates and side-by-side
structures are formed (Moens & Rapport, 1971; Zickler & Olson, 1975). Then, the parental
and daughter spindle pole bodies separate and the second meiotic spindle extends between the
two (Moens & Rapport, 1971; Zickler & Olson, 1975). Considering these processes, we infer
that in mutant SOS4 the pre-first meiotic spindle pole body duplication is normal but the
pre-second meiotic duplications are so defective at 29 OC that newly synthesized spindle pole
bodies lack the outer plaques (Fig. 5). This means that duplication of outer plaques at the
pre-first meiotic division was not affected at 29 OC by the hfdl-1 mutation, but those at the
pre-second meiotic division were affected.
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Fig. 1. Electron micrographs of a second meiotic spindle of strain MT13 (HFDZ+/HFDl+)
in two serial sections. (a) A spindle pole body
having normal inner (IP) and outer (OP) plaques, with microtubules (MT)extending from it. (b) A spindle pole body at the opposite pole,
again having normal inner and outer plaques. The bar markers represent 0.2 p.
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S . OKAMOTO A N D T. IINO
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Meiosis in S. cerevisiae
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Fig. 2. Electron micrographs of a second meiotic spindle in mutant SOS4 (hfdl-Zlhfdl-I).One of
the poles has a spindle pole body having normal inner (IP) and outer (OP) plaques, while the other
has a spindle pole body entirely lacking the outer plaque. (a) The spindle at stage I, with no prospore
wall membrane at either pole. (6)The spindle at stage 11, with a normal prospore wall membrane only at
the pole having the outer plaque (also see Fig. 3). The bar markers represent 0.2 prn in ( a ) and 0.5 pm
in (b).
Davidow et al. (1980) reported that under certain physiological conditions, presumably of
nutrient deprivation, strain AP-1 of S. cerevisiae manifested an Hfd phenotype and formed
non-sister spindle pole bodies entirely lacking outer plaque at the second meiotic division
(Davidow et al., 1980). These phenotypes are similar to those of the hfdl-1 mutant. However,
no prospore wall membrane was detected in AP-1 near the pole lacking the outer plaque
(Davidow et al., 1980), while an abnormal one was detected at this pole in the hfdl-I mutant.
There are three possible sequences of events which might cause the phenotype of the hfdl -I
mutation. The first is that the hfdl-1 mutation blocks the morphogenesis of outer plaques at
the pre-second meiotic division and the absence of outer plaques results in the development of
abnormal prospore wall membranes. The second is that the development of prospore wall
membranes is blocked by the hfdl-I mutation and the resulting abnormalities lead to the
absence of outer plaques. The third is that the hfdl-1 mutation causes both abnormalities in
parallel. Since morphogenesis of outer plaques at the pre-second meiotic division always
precedes the development of prospore wall membranes in wild-type strains (Moens &
Rapport, 1971; Horesh et al., 1979), the second possibility is unlikely. The results reported
here do not discriminate between the remaining two possibilities.
The observed structures of 17 stage I1 spindle poles of MT 13 or SOS4 cells can be divided
into four classes as follows (numbers in parentheses represent the observed numbers of poles
belonging to that class): 1 - a pole which has full length outer plaque and a normal prospore
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S. O K A M O T O A N D T . I I N O
Fig. 3. Electron micrographs of the spindle pole bodies in Fig. 2 (b) at higher magnification. (a, b) Two
serial sections of the spindle pole body having the outer plaque (OP), with a normal prospore wall
membrane (PSW) near the pole. (c, d)Two serial sections of the opposite spindle pole body lacking the
outer plaque, with an abnormal prospore wall membrane (APSW) near the pole. The bar markers
represent 0-2 pm.
wall membrane (11); 2 - a pole which has no detectable outer plaque and an abnormal
prospore wall membrane (4); 3 - a pole which has a smaller and thinner outer plaque than
normal and a normal prospore wall membrane (1); 4 - a pole which has a much smaller and
thinner outer plaque than that described in class 3 and has an abnormal prospore wall
membrane (1).
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Fig. 4. Electron micrograph of a second meiotic cell in mutant SOS4. A spindle pole body (arrow) which lacks the outer plaque has an
abnormal prospore wall membrane (APSW) near the pole. Double arrows indicate the location of the opposite spindle pole body (which
appeared in subsequent sections), with a normal prospore wall membrane (PSW) developing near the pole. The bar markers represent
0.1 pm.
w
CI
cn
c
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S . OKAMOTO A N D T. IINO
’5
Fig. 5. Schematic representation of meiosis and ascopore formation in mutant SOS4 (hfdl-llhfdl-I) at
29 OC. The intermediate figure for spindle formation shown in parenthesis has not been detected in
meiosis but has been detected in mitosis (Peterson & Ris, 1976). Cell walls are omitted. 1, Nuclear
envelope; 2, inner plaque; 3, outer plaque; 4, prospore wall membrane; 5, abnormal prospore wall
membrane.
Poles belonging to class 1 have been observed by many workers and a role for the outer
plaque in the formation of the prospore wall membrane has been suggested (Moens &
Rapport, 1971; Zickler & Olson, 1975; Horesh et al., 1979; Davidow et al., 1980). Poles
belonging to classes 2, 3 and 4 have not been reported previously. The presence of these
classes suggests that the full length of the outer plaque is not a prerequisite for the formation
of a prospore wall membrane. Moreover, it indicates that in the absence of the outer plaque,
formation of the prospore wall membrane can proceed partially.
We are grateful to D r K. Tanaka, Nagoya University, for advising one of us (S.O.) on the method of
preparation of blocks for electron microscopy, and providing us with the accelerator S- 1 for Spurr’s resin.
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