1 Department of Molecular Genetics, Research Institute for Microbial Diseases,
Osaka University, 3-1 Yamadaoka, Suita, Osaka 565-0871, Japan
2 Department of Integrated Biosciences, Graduate School of Frontier Sciences
Tokyo University, Bldg. FSB-101/601, 5-1-5 Kashiwanoha, Kashiwa, Chiba
277-8562, Japan
Author for correspondence (e-mail:
hnojima{at}biken.osaka-u.ac.jp)
Accepted 18 March 2003
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Summary |
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Key words: Meiosi, Spore formation, Forespore membrane, Microtubule, Spindle pole body, Schizosaccharomyces pombe
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Introduction |
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Spore formation is initiated during meiosis II in that during this
division, the dividing nuclei start to be surrounded by a double unit
membrane, called the forespore membrane. A previous study has shown that the
forespore membrane develops near the outer plaques of the spindle pole body
(SPB) during meiosis II and plays an important role in the packaging of the
haploid nucleus (Hirata and Shimoda,
1994) However, the exact molecular mechanism driving forespore
membrane development is unclear.
For efficient spore production, the formation of the forespore membrane
must be coordinated with the behavior of the dividing nuclei during meiosis
II. The SPB is thought to be a key structure that links these two events. The
SPB may act similarly to the mammalian centrosome, which functions as a
microtubule-organizing center (MTOC). The SPB enters and leaves the nuclear
envelope during the cell cycle (Ding et
al., 1997), and has been observed by immunostaining to change from
a dot-like image into a crescent during meiosis II
(Hirata and Shimoda, 1994
;
Hagan and Yanagida, 1995
). The
correct morphological alteration of the SPB and the resulting normal
sporulation depends on Spo15, an SPB component that is essential for forespore
membrane formation (Ikemoto et al.,
2000
). Electron microscopic observation has revealed that the
forespore membrane is initially assembled next to the SPB, which changes
morphologically into a multilayered structure during meiotic division II
(Tanaka and Hirata, 1982
;
Hirata and Shimoda, 1994
;
Hagan and Yanagida, 1995
). This
suggests that SPB modification is required to initiate forespore membrane
formation. Thus, SPBs play an additional role in sporulation that is distinct
from their known roles as MTOC scaffolds in mitosis and meiotic division
I.
Other molecules that appear to be required for normal sporulation are
Spo20, an S. pombe phosphatidylinositol-transfer protein
(Nakase et al., 2001), Spo3, a
forespore membrane component (Nakamura et
al., 2001
), and Meu10, a molecule with no homology to
well-characterized proteins (Tougan et
al., 2001
). Thus, sporulation appears to consist of several
cytologically distinct steps that include the modification of SPBs, the
assembly of forespore membranes around the SPBs, the enclosure of nuclei by
the forespore membranes, the maturation of the spore wall, and the autolysis
of the ascus wall. With regard to the forespore membrane, it is assembled by
the fusion of vesicles perhaps derived from the endoplasmic reticulum (ER)
and/or the Golgi apparatus (Tanaka and
Hirata, 1982
; Hirata and
Shimoda, 1994
). However, little is known about how the forespore
membrane assembles in fission yeast, which is in contrast to the prolific
studies examining the same phenomenon in budding yeast (for a review, see
Pringle et al., 1997
).
To understand how meiosis and sporulation are regulated in S.
pombe in more detail, we have established a subtracted cDNA library to
comprehensively isolate meiosis/sporulation-specific genes that we have
denoted as meu (meiotic expression
upregulated) (Watanabe et al.,
2001). Previously, we reported that the gene product generated
from one of these genes, meu13+, promotes homologous pairing
independently of homologous recombination and regulates the meiotic
recombination checkpoint (Nabeshima et
al., 2001
; Shimada et al.,
2002
). Furthermore, we found that the meu10+ product is
required for ascospore maturation (Tougan
et al., 2001
). Here, we characterize another meu gene,
meu14+. We show that the S. pombe Meu14 protein is
expressed specifically during sporulation and appears to play a role in the
extension of the forespore membrane and perhaps also in the function of the
SPB and/or the meiotic spindles.
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Materials and Methods |
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Isolation and deletion of meu14+
The genomic library (Fukushima et al.,
2000) was made using partial Sau3AI DNA fragments
inserted into the BamHI site of the Bluescript KS(+) vector
(Stratagene, La Jolla, CA). Colony hybridization with the
meu14+ cDNA fragment as a probe allowed us to clone the
genomic fragment containing the meu14+ gene from this
library. To disrupt meu14+, a 1.1 kb NspI
fragment carrying the meu14+ genomic DNA was replaced with
the 1.8 kb HindIII fragment containing ura4+
using a synthetic linker. The meu14+::ura4+ DNA
fragment digested with KpnI (-1104 bp site from start codon) and
PstI (1050 bp site from stop codon) was used to transform wild-type
cells (TP4D-5A/TP4D-1D). This procedure removed all of the coding region of
Meu14, including the initiation codon. The Ura+ transformants were
then screened for the disruption of one of the meu14+ gene
copies by genomic Southern blot analysis. Tetrads from these cells were then
dissected. Northern and Southern blot analyses were performed as described
previously (Watanabe et al.,
2001
).
Preparation of Meu14-GFP
The Meu14 protein fused C-terminally with the green fluorescence protein
(GFP) was made by first synthesizing oligonucleotides FN
(5'-GGCGCGCCGCATATGGGCACTCAACCATCTTAC-3') and FC
(5'-GCGGCCGCGGCAAGAAAACAGTGGATTTTGC-3'), which
correspond to the initiation and termination sites of Meu14, respectively. The
NdeI and NotI sites introduced into the oligonucleotides are
underlined, respectively. Using these oligonucleotides as primers and
meu14+ genomic DNA as a template, we performed a
polymerase chain reaction (PCR) and generated a meu14+
fragment harboring the NdeI and NotI sites in the N- and
C-termini, respectively. After performing a TA-cloning procedure, this DNA
fragment was digested by NdeI and NotI and inserted into the
pRGT1 vector (a gift from M. Yamamoto, University of Tokyo), which was
designed to fuse GFP to the insert if the DNA was inserted in-frame via
NdeI/NotI sites using the pREP1 vector
(Maundrell, 1993). For
Meu14-GFP protein expression during vegetative growth, the
meu14+ DNA fragment generated by PCR to contain
NdeI and NotI sites at the ends was inserted into pRGT81, a
modified pRGT1 vector designed to carry a weak nmt promoter. This
construct is termed pRGT81-meu14+. To obtain the 5'
upstream promoter region of meu14+, we performed PCR using
the primers 5N (5'-CTGCAGATCCGAGCAAGAAGAGGCT-3') and 5C
(5'-CATATGGATTGTTTACG TTTCAGA-3'). The PstI
and NdeI sites introduced into the oligonucleotides are underlined,
respectively. The nmt1 promoter between the PstI and
NdeI sites in the pRGT1-meu14+ construct was
replaced by this amplified DNA fragment. We picked four independent clones and
determined the DNA sequences of the amplified regions in order to select the
clone whose sequence does not contain point mutations introduced by the PCR
amplification. This construct is termed pNP-meu14+. Next,
the NspI genomic DNA fragment corresponding to the 3'
downstream region of meu14+ (+61 to 1138 bp from stop
codon) was inserted into the SmaI site between the GFP gene and the
nmt1 terminator in the pNP-meu14+ construct. This
meu14+-gfp DNA fragment carrying about 1 kb of both
upstream and downstream regions was digested with PstI and
KpnI, and then used to transform
meu14+::ura4+ cells (YDO100). The
Ura- transformants were obtained by screening for the clone that
survived in the medium containing 5-Fruoroorotic Acid (5-FOA), which was
confirmed by Southern blot analysis (data not shown).
Immunofluorescence
Meiotic cells were fixed following the procedure of Hagan and Hyams using
glutaraldehyde and paraformaldehyde (Hagan
and Hyams, 1988). In indirect immunofluorescence microscopy
(Hagan and Yanagida, 1995
), the
SPB was stained with the anti-Sad1 antibody (a gift from M. Yanagida,
University of Kyoto), microtubules were stained with the TAT-1 antibody [a
gift from K. Gull, University of Manchester
(Woods et al., 1989
)], and the
nuclear pore complex was stained with the MAb414 antibody (CRP, Denver, PA).
Spo3-HA (a gift from C. Shimoda, City University of Osaka) was stained with
the anti-HA antibody 3F10 (Boehringer Mannheim, Mannheim, Germany).
Subsequently, a Texas-Red-conjugated sheep anti-mouse antibody (Amersham
Biosciences, Piscataway, NJ) was used to visualize the microtubules, Spo3-HA,
and the nuclear pore complex. An Alexa-488 or 594-conjugated goat anti-rabbit
antibody (Molecular Probes, Eugene, OR), a Cy5-conjugated donkey anti-rabbit
antibody (Jackson ImmunoResearch Laboratories, West Grove, PA), and a
Texas-Red-conjugated donkey anti-rabbit antibody (Amersham Biosciences,
Piscataway, NJ) were used to stain Sad1. The samples were then stained with
0.2 mg/ml DAPI or Hoechst 33342 in PBS for 5 minutes and mounted with
antifade-containing Vectashield mounting medium (Vector Laboratories,
Burlingame, CA). Fluorescence images of these cells were observed using a
fluorescence microscope (Axiophot, Zeiss, Germany or BX51, Olympus, Tokyo,
Japan) with a charge-coupled device (CCD) cameras (Photometrics PXL1400) or
Cool SNAP CCD camera (Roper Scientific, Sad Diego, CA). Immunofluorescence
images were acquired using Adobe PhotoShop 6.0.
Fluorescence in vivo imaging of Meu14 and chromosomes
To stain chromosomes and detect Meu14-GFP in living meiotic cells,
meu14+-gfp cells (YDO120) were first transferred to EMM2-N
to induce meiosis. The meiotic cells were then stained with Hoechst 33342 (1.0
µg/ml) for a few minutes. Stained live cells were mounted on a coverslip by
spotting, and microscopic observations were carried out with the Delta Vision
microscope system (Applied Precision, Issaquah, WA). Images were taken with a
0.2 second exposure at 2 minute intervals. This system allows multi-color and
three-dimensional acquisition of digitized images with a cooled CCD camera.
The images of the live meiotic cells stained with Hoechst 33342 were obtained
as described (Chikashige et al.,
1994).
Preparation of cell extracts and immunoblotting
S. pombe cells (3.3x108 cells) were suspended in
0.3 ml of HB buffer (25 mM MOPS, pH 7.2, 15 mM MgCl2, 15 mM EGTA,
60 mM ß-glycerophosphate, 15 mM p-nitrophe-nylphosphate, 0.1 mM
Na3VO4, 1 mM dithiothreitol, 1 mM phenylmethylsulf
o-nylfluoride, and 20 mg each of leupeptin and aprotinin per ml) and disrupted
with acid-washed glass beads using a bead beater. Proteins in the extracts
were separated by SDS-PAGE and transferred onto polyvinylidene difluoride
membranes (Immobilon; Millipore, Bedford, MA). Blots were probed with the rat
anti-GFP antibody (a gift from S. Fujita of Mitsubishi Kagaku Institute of
Life Sciences, Tokyo, Japan) and the rabbit anti-Cdc2 antibody (Santa Cruz
Biotechnology, Santa Cruz, CA). The bands bound by the horseradish
peroxidase-conjugated goat anti-rat IgG (Santa Cruz Biotechnology, Santa Cruz,
CA) for the anti-GFP antibody or goat anti-mouse IgG (ICN Pharmaceuticals,
Costa Mesa, CA) for the anti-Cdc2 antibody were visualized using the
Renaissance system (NEN Life Sciences, Boston, MA).
Electron microscopic observations
Wild-type diploid (TP4D-5A/TP4D-1D) and meu14 diploid
(YDO110) cells were grown in EMM2 medium at 33°C, transferred to EMM-N
medium at 30°C, and harvested by centrifugation 6 or 8 hours later. The
pelleted cells were cryofixed by high-pressure-freezing using a HPM 010
(Bal-Tec, Balzers, Liechtenstein) as previously described
(Humbel et al., 2001
) before
being subjected to electron microscopy (Hitachi H-7600, Hitachi
High-Technologies, Tokyo, Japan).
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Results |
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Furthermore, we investigated whether meu14+ gene expression
occurs under the control of the meiosis-specific transcription regulator Mei4
because the Mei4 target sequence termed FLEX (GTAAACAAACAGA) occurs in the
5' upstream region of meu14+
(Horie et al., 1998;
Abe and Shimoda, 2000
).
Northern blot analysis showed that meu14+ gene expression was not
detectable in the mei4
strain (Fig.
1B, right panel). This suggests that meu14+ expression
is under the direct control of Mei4.
Structural characterization of the meu14+ gene and its
product
We used the isolated meu14+ cDNA fragment as a probe to
screen the genomic library of h- L972 cells
(Fukushima et al., 2000). This
allowed us to clone a DNA fragment that included the surrounding regions of
the meu14+ gene, and the DNA sequence was determined (DDBJ
accession No. AB016983). Comparison of the genomic DNA and cDNA sequences
indicated that the meu14+ gene contains one intron-bearing
consensus intron splice and branch sequences (data not shown).
meu14+ encodes a predicted translation product consisting
of 335 amino acids with a molecular weight of
37 kDa. The PSORT II server
program
(http://psort.ims.u-tokyo.ac.jp/)
was used to search for motifs and revealed that the Meu14 protein has a
predicted coiled-coil (C-C) motif (Burkhard
et al., 2001
) in the central region of the molecule
(Fig. 1Ci).
The BLAST algorism (http://www.genome.ad.jp/) was used to search for homologous genes and revealed that there are two genes in S. pombe that are paralogous to meu14+. We have denoted them as mfp1+ (SPAC3C7.02C) and mfp2+ (SPCC736.15) after meu fourteen paralog. Notably, an intimate homolog of meu14+ does not exist in the genome of S. cerevisiae. Maximum matching alignment (Fig. 1Cii) and a phylogenetic tree (Fig. 1D) based on the overall amino acid sequences of the proteins clearly show that the Mfp1 and Mfp2 proteins are more similar to the budding yeast homologues Ygr086c and Yp1004c than to Meu14. Genes with obvious homology to meu14+ have not been identified in any other organism.
Northern blot analysis indicated that, like meu14+, mfp1+ transcription is only induced during the meiosis/sporulation process, although it was expressed later (8-12 hours; Fig. 1A). It is strange, however, that when the pat1-114 strain is used, mfp1+ displays two peaks (Fig. 1B). We obtained this result repeatedly using different northern blots. The reason for this is currently under investigation. Nevertheless, when we constructed a null mutation of mfp1+, abnormal phenotypes in vegetative growth or the meiosis/sporulation of diploid cells were not observed (data not shown). With regard to mfp2+, northern blot analysis revealed that it is transcribed both during vegetative growth (0 hours) and meiosis (Fig. 1A). Moreover, neither mfp1+ nor mfp2+ seems to be under the control of Mie4 transcription factor (Fig. 1B, right panel). Thus, mfp1+ and mfp2+ genes were not studied further.
meu14cells fail to sporulate and produce a high
frequency of abnormal tetranucleated cells
To assess the physiological role of meu14+, a deletion mutant
that expresses no Meu14 protein was constructed by one-step gene replacement
(see Materials and Methods). We confirmed the successful disruption of
meu14+ by examining the transformants for the presence or absence
of the uracil auxotrophic marker and by Southern analysis.
Diploid cells in which one of the meu14+ genes had been
replaced by ura4+ were sporulated and germinated. The
segregation ratio compared to the wild-type was 1:1. All of the resulting
spores were viable, indicating that the meu14+ gene is not
essential for vegetative growth. The growth properties and the cell size and
morphology of meu14 cells were also indistinguishable from
those of the wild-type cells. Thus, we conclude that the
meu14+ gene has no obvious function in the vegetative
growth phase.
FACS analysis indicated that premeiotic DNA synthesis was normal in
meu14 cells (data not shown). Spore walls were not formed by diploid
cells homozygous for the meu14
gene
(Fig. 2A,B), indicating that
meu14
cells are defective in sporulation. Progression through the
various meiotic steps was monitored by counting the frequency of various
numbers of nuclei at 2 hour intervals by fluorescence microscopy and showed
that meu14
cells proceed normally through the onset of the two meiotic
divisions, as its kinetics are comparable to that of the parental wild-type
control (Fig. 2C). Thus, the
onset of the nuclear divisions occurring during meiosis I and II is normal in
meu14
cells. However, at 12 hours, at which point 80% of the wild-type
cells had undergone sporulation, no asci were observed in meu14
cells,
confirming sporulation in meu14
cells is severely impaired. Notably,
horse-tail phase seems longer in meu14
cells than wild-type, which
suggests that Meu14 may also play a role at this phase. The meu14
cells
produced abnormal tetranucleate cells at a high frequency
(Fig. 2D). We could classify
these abnormal tetranucleate cells into five classes (see legend to
Fig. 2D). Taken together, we
conclude that meu14
cells become abnormal after meiosis II has
commenced.
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SPB assembly is aberrant in meu14 cells during meiosis II
To determine if the abnormal tetranucleate cells of meu14
strain are due to the aberrant function of the microtubules or the SPBs,
meu14
cells were induced to enter meiosis. The cell
populations enriched in anaphase II cells were then stained by Hoechst 33342
to reveal the nuclei and immunostained with the anti--tubulin antibody (TAT-1)
and the anti-Sad1 antibody to delineate the microtubules and the SPBs
(Hagan and Yanagida, 1995
),
respectively. We found that meu14
cells are normal at meiosis
I (Fig. 3Av,vi). However, many
of the meu14
cells progressing though meiosis II contained
fragmented or unequally-shaped microtubule bundles
(Fig. 3Ai-iv). Moreover,
multiple Sad1-stained objects were observed more frequently in
meu14
cells (Fig.
3A)
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To confirm that the anti-Sad1 antibody specifically recognize the SPBs in
meu14 cells, we induced cells that express the Spo15 protein fused to
GFP to enter meiosis II and then stained them with Hoechst 33342 and the
anti-Sad1 antibody. Spo15 is a SPB component that is required for SPB
alteration in Meiosis II and sporulation
(Ikemoto et al., 2000
). The
abnormal Sad1 signals colocalized with Spo15-GFP fluorescence signals
(Fig. 3B). Thus, the
organization or segregation of the SPBs in meu14
cells is abnormal.
This suggests that the SPBs may not be able to act properly as scaffolds for
forespore membrane formation, which may explain the lack of spore formation in
meu14
cells. Supporting this is that the meu14
cells lack the
crescent form of SPBs, which resembles the abnormality observed in
spo15
cells (Ikemoto et al.,
2000
). Thus, the abnormal spore formation in meu14
cells
may be due to a failure in the formation or segregation of SPBs during meiosis
II.
Subcellular localization of Meu14-GFP during meiosis
To assess the behavior of the endogenous Meu14 protein during meiosis, we
constructed a strain in which meu14+ was replaced by a
meu14+-gfp gene that is designed to express the Meu14 protein fused
with GFP at its C-terminal end. Since expression of this meu14+-gfp
gene is controlled by the native meu14+ promoter, its expression is
expected to be identical to that of the intact meu14+ gene. That
the meu14+-gfp gene could fully complement the meiotic defects in
meu14 diploid cells, and that these cells generated normal ascospores
implies that the Meu14-GFP protein retains its function (data not shown).
Western blot analysis using cell lysates reveals that the expressed Meu14-GFP
protein migrates at the expected size, indicating it is not degraded in vivo
(Fig. 4A). Western blotting
also showed it is expressed only during meiosis II (4 to 8 hours)
(Fig. 4A).
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Cells carrying meu14+-gfp were induced to enter meiosis and were fixed before fluorescence analysis of DNA, Meu14-GFP, and tubulin. The timing of meiotic progression in the meu14+-gfp cells was confirmed to be quite similar to that of wild-type cells (data not shown). Based on the number of nuclei and the morphology of the microtubules, we judged the stage of meiosis of the individual cells and collected typical images to represent each stage of meiosis. Fluorescence signals from Meu14-GFP were not detected in mitotically growing cells or in early meiotic cells (at interphase, horse-tail, or meiosis I; Fig. 4B). The Meu14-GFP signal first appeared at prometaphase II as a blur in both the nucleus and the cytoplasm. After this, four strong rings appeared in the cells at metaphase II, and these four rings became bigger at anaphase II. The rings shrank at post-anaphase II and were again visualized as four small rings.
When we measured the distances between the two Meu14-GFP rings and plotted these versus the nuclear distance (Fig. 4C; upper right panel) or the length of microtubules (lower right panel), we found that the curves are similar, indicating that the Meu14-GFP rings behave in good coordination with both nuclear division and microtubule elongation. By counting the number of cells displaying Meu14-GFP signals, we found that Meu14-GFP was detectable in 78% of short-spindle cells at prometaphase II, in 100% of elongated-spindle cells at anaphase II, and in 48% of spindle-negative cells at post-anaphase II.
Immunostaining at various stages of meiosis also revealed that Meu14-GFP
localizes next to the Sad1 signal on the SPB only at metaphase II
(Fig. 4D; white arrow). This
suggests that Meu14-GFP is located on the cytoplasmic side of the SPB. When
the nuclear pore complex (NPC) was immunostained with the mAB414 antibody
(Wilkinson et al., 2000),
Meu14-GFP was detected outside of the NPC at late meiosis II
(Fig. 4E).
Localization of Meu14-GFP in live cells during meiosis
The behavior of Meu14-GFP during meiosis suggests that it is transiently
expressed and then degraded (Fig.
1B, Fig. 4A), and
that before its degradation it moves around in the cell. To further
investigate this dynamic behavior of Meu14-GFP, we observed the protein in
live meu14+-gfp cells that were induced to enter meiosis and then
stained with Hoechst 33342 to visualize the nucleus. The images have been
stored as a file that can be run as an animation available on the internet. We
show in Fig. 5 several time
lapse images taken at 6 minute intervals of a cell passing from prophase II to
metaphase II (Fig. 5A; see
Movies 1 and 2,
http://jcs.biologists.org/supplemental)
and of a cell progressing from metaphase II to anaphase II
(Fig. 5B; see Movies 3 and
4).
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Meu14-GFP first appears at early meiosis II as a dot at the periphery of each of the two nuclei. The dots then duplicate and move around the nuclear membrane (as indicated by white arrowheads in Fig. 5Ai,ii). This behavior of Meu14-GFP resembles that of the SPB. Thereafter, the Meu14-GFP signals start to increase (time 0 in Fig. 5A) from the opposite ends of the two nuclei. Each nucleus develops small ring-shaped structures at either end. These structures gradually increase their diameters in a synchronized manner. The two rings approach each other until their diameters are maximized, after which they start to separate because of the nuclear division of meiosis II. Subsequently, as the nuclei separate from each other, the diameters of the two rings gradually shrink until they become two dots in the center of the two dividing nuclei (Fig. 5B). In post-anaphase II, the strengths of the Meu14-GFP ring/dot signals gradually diminish and the Meu14-GFP signal is observed to be diffusely distributed among the four spores. The diminished Meu14-GFP signal just before the maturation of the ascospores coincides with the disappearance of the Meu14-GFP band in immunoblotting (Fig. 4A).
At the late stage of meiosis II, the dim stain throughout the nuclear region almost completely disappears and only rings are observed, which is shown in Fig. 5Aii and Fig. 5Bii. The pictures are colorless so as to highlight the GFP signals. These pictures suggest that Meu14 localizes in the nuclear region and then assembles into rings during meiosis II.
When the Meu14-GFP rings are closely examined by an enlarged view rotated on the Y-axis by an angle of seventy-two degrees to obtain three-dimensional images, they are found to form a structure like 'assembled beads' (Fig. 5C, Movie 5). We counted more than ten beads that composed each Meu14-GFP ring in these images. Notably, at the end of meiosis II, when the strong Meu14-GFP signal is observed as an intense dot at the edge of the nucleus (Fig. 5B), the weak Meu14-GFP signal was also observed to have a balloon-like shape (Fig. 5D). This is probably the forespore membrane, indicating that some portion of Meu14-GFP is distributed at the forespore membrane.
Meu14 is required for the extension of the forespore membrane from
the SPBs
To investigate whether Meu14 is located on the forespore membrane, we
compared the localization of Meu14-GFP to that of the Spo3-HA molecule, which
is a forespore membrane component
(Nakamura et al., 2001).
Spo3-HA fluorescence signals appeared as two semicircular structures that
enclose the nucleus at metaphase II, and as four capsular structures enclosing
the nucleus at anaphase II (Fig.
6A). The SPBs were situated at the center of each semicircle
displayed by Spo3-HA. At the edges of these semicircles and capsules,
Meu14-GFP could be detected. Thus, Meu14-GFP occurs at the leading edge of the
forespore membrane during meiosis II. The fluorescence of Meu14-GFP that
localized at the edge of the forespore membrane became reduced as the edge of
the forespore membrane encloses itself, while Spo3, the component of the
forespore membrane, remains until the forespore membrane growth is completed
(Fig. 6Ai-v)
(Nakamura et al., 2001
). The
edge of the extended forespore membrane appears to play an important step for
the mature spores.
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Next, we examined the localization of Psyl-GFP in meu14 cells by
fluorescence microscopy. Psyl, like Spo3, also serves as a marker for the
formation of the forespore membrane
(Nakamura et al., 2001
). The
reason for using the Psy1-GFP is that fluorescence of Psy1-GFP can be detected
much more easily during fluorescence microscopy than the fluorescence of
Spo3-HA (data not shown). Psy1-GFP was integrated into the genome of
meu14
cells and wild-type cells. The cells were then induced to enter
meiosis II and stained with the anti-Sad1 antibody to mark the SPBs
(Fig. 6B). In wild-type cells,
the forespore membranes visualized by Psy1-GFP assembled normally as a circle
and at meiosis II enclosed the haploid nucleus stained by Hoechst 33342
(Fig. 6Bi). In wild-type cells,
the forespore membrane extends symmetrically from the SPB and results in this
circular structure [data not shown
(Nakamura et al., 2001
). Here,
the position of SPBs indicates the orientation of the nuclear division at
meiosis II. However, in most of the meu14
cells, the semicircular
forespore membrane is formed in an inappropriate place, thus failing to
properly encircle the nucleus (Fig.
6Bii-iv). In meu14
cells, only few of the forespore
membrane leading edges were normally assembled as in wild-type cells
(Fig. 6C). The majority were
abnormally assembled. These observations indicate that Meu14 is required for
the proper extension of forespore membrane from the SPBs. As shown in
Fig. 6C, the meu14
ascospores were abnormal in number and shape. In contrast, in wild-type cells,
the forespore membrane encapsulated each haploid nucleus (>90%), while the
initiation of the forespore membrane in the meu14
cells is normal, most
later fail to develop a normal morphology.
Meu14-GFP can form ring shapes in the absence of Spo3 or Spo15
Previous reports show that spo15+ is required for the
alternation of the SPBs in meiosis II and spo3+ is required for the
accurate formation of the forespore
(Ikemoto et al., 2000;
Nakamura et al., 2001
). To
examine if Spo3 and Spo15 are required for the localization of Meu14-GFP at
the edge of forespore membrane, we constructed the spo3
meu14+-gfp and spo15
meu14+-gfp strains
(Fig. 6D). The
h90spo3
meu14+-gfp or h90 spo15
meu14+-gfp strains were induced to proceed with zygotic meiosis and
sporulation by nitrogen starvation, after which the localization of Meu14-GFP
was visualized by fluorescence microscopy. As shown in
Fig. 6Di, Meu14-GFP was
detected at the edge of the forespore membrane in zygotic meiosis as also
observed in azygotic meiosis (Fig.
4D). The fluorescence of Meu14-GFP was found to form ring shapes
in both spo3
and spo15
. The four ring shapes of Meu14-GFP showed
a synchronized action in spo3
but not in spo15
. In spo3
cells, we found that Meu14 is situated at the edge of a spore-like body
(Fig. 6Diii,iv). In many
spo15
cells, Meu14 was undetectable
(Fig. 6Dv) while in others, the
number, size and position of Meu14 signals were abnormal
(Fig. 6Dvi,vii). Thus,
Meu14-GFP is able to localize at the edge of the abnormal forespore membrane
in the absence of Spo3 and Spo15.
Morphology observed by electron microscopy
To investigate the structure of the forespore membrane and the spore wall
of meu14 cells in more detail, we examined their morphology by
thin-section electron microscopy (EM). We treated the cells by high-pressure
freezing (Humbel et al., 2001
)
to minimize the artifacts that can arise during the pre-fixation process. The
substituted cells were then fixed and observed by electron microscopy. From
the meu14
cells, we obtained images showing that the forespore
membranes and spore wall failed to form accurately and displayed abnormal
morphology (Fig. 7Ai) as
compared to those of wild-type cells (Fig.
7Aii). Enlarged pictures show some of the various abnormalities of
the meu14
spore wall observed, such as a small spore-like
structure without a nucleus (iii), an incompletely encapsulated nucleus (iv),
and a nucleus without a spore wall (v). Furthermore, in meu14
cells, the thickness of the spore wall is not homogeneous compared to that of
the wild-type cells (vi).
|
EM images also show that the interaction of the forespore membrane (black
arrows) with the SPB (white arrows) becomes abnormal in meu14
cells (Fig. 7Bi). That is, the
forespore membrane segregates from the SPB, thus extending abnormally to the
opposite direction to the nucleus (ii). The relative orientation of the
forespore membrane with the SPB (iii) is distinct from that of wild-type cells
(iv). This abnormal segregation of the forespore membrane from SPB is
consistent with our indirect immunofluorescence data
(Fig. 6B). Some
meu14
cells showed abnormally shaped SPBs as indicated by
white arrows in Fig. 7C. In
such cases, encapsulation of the nucleus by the forespore membrane is mostly
unsuccessful (i and iv). Enlarged pictures display the abnormally enclosed
forespore membrane without a nucleus (ii) next to the naked nucleus (iii),
both of which carry improperly formed SPBs. Moreover, some of the SPBs do not
localize between the nucleus and forespore membrane but rather, are situated
outside of these structures (v and vi).
|
The organization of the spindles are also abnormal in meu14
cells (Fig. 7D), as some cells
have abnormally bundled spindle-like structures (i) that are improperly
enclosed by the abnormally formed forespore membranes. Some
meu14
cells showed seemingly normal spindle formation (ii-iv),
but enclosure of the bundle is abnormal when no SPB is found (iii). It is also
unusual that a spindle-like structure is observed in the spores, indicating
that the handling of the spindles is also abnormal in meu14
cells (Fig. 7Av).
![]() |
Discussion |
---|
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---|
During meiosis II, Meu14-GFP is found first at the cytoplasmic side of the SPB (Fig. 4D) and then at the leading edge of the forespore membrane (Fig. 6A). Time-lapse microscopic analysis using a meu14+-gfp strain (Fig. 5A,B) allowed us to observe the dynamic behavior of Meu14-GFP in live cells during meiosis II. Meu14-GFP first appears as a dot at the periphery of the nucleus, where it is duplicated and then moves to either end of the nucleus to form a pair of rings. The rings gradually increase in diameter. At the same time, the forespore membrane, which grows from the SPB, also increases. After the forespore membrane completely encloses the haploid nucleus at the end of meiosis II, the diameter of the rings gradually shrink until they appear as dots, after which they disappear. Thus, Meu14 is required specifically at meiosis II, where it seems important for several processes: (i) it guides the formation of the forespore membrane as it develops during meiosis II and sporulation, and (ii) it assists the equal partitioning of the nucleus and the temporal coordination of this event with chromosome segregation during meiosis II.
Many studies have been conducted to comprehensively identify and
characterize the subcellular localization of yeast proteins involved in
meiosis and mitosis (Sawin and Nurse,
1996; Ding et al.,
2000
). Of note is that none of these studies identified Meu14.
Thus, the subcellular localization of Meu14-GFP during S. pombe
meiosis is unique.
Loss of Meu14 may destabilize the SPB
The dynamic movement of Meu14 at meiosis II indicates that Meu14 is a novel
type of molecule that regulates the architecture of cellular structures by
using the SPB as one of its scaffolds. The SPB is a multilayered proteinaceous
structure that is inserted into the nuclear envelope in S. pombe
(Hirata and Tanaka, 1982;
Ding et al., 1997
). It is
thought that the SPB buried in the nuclear membrane is stable due to astral
microtubule interaction with the cell cortex. In the second meiotic nuclear
division, the astral microtubules on the cytoplasmic side are exchanged with a
multilayered SPB structure from which the forespore membrane, whose analogue
in S. cerevisiae is the prospore membrane
(Neiman, 1998
), extends to
enclose the spore nucleus. Thus, during meiosis II, astral microtubules are
absent. We believe that the lack of Meu14 causes the SPB buried in the nuclear
membrane to become unstable, which would explain the fact that
meu14
cells display deformed and multiple SPBs that are not
embedded in the nuclear envelope during meiosis II
(Fig. 7C). This also explains
our observation that the SPBs in meu14
cells, when observed by
Spo15-GFP, a protein that regulates the onset of forespore membrane extension
(Ikemoto et al., 2000
), are
abnormally organized or segregated. It is likely that Meu14 also plays a role
in the structure of meiosis I SPB, possibly during anaphase I in preparation
for meiosis II. Thus, Meu14 may already associate with the meiosis I SPBs, but
it cannot be detected because Meu14-GFP at this stage is too faint to be
recognized as GFP signals (Fig.
4B).
Meu14 guides the formation of the forespore membrane
Recently, Spo3 and Psy1 of S. pombe and Don1, Ady3, and Ssp1 of
S. cerevisiae (Knop and Strasser,
2000; Moreno-Borchart et al.,
2001
; Nickas and Neiman,
2002
) were identified to be protein components of the forespore
membrane. All have been shown to be localized at the cytoplasmic side of SPB.
We used a Psy1-GFP integrated cell to trace the assembly and extension of the
forespore membrane in meu14
cells that were also stained with markers
for the nucleus and SPBs. We observed that the forespore membrane had an
abnormally assembled leading edge that could not extend out from the SPB and
thus could not properly encircle the nucleus in most meu14
cells
(Fig. 6B). We also observed
that the loss of meu14+, as in the spo3 mutant, caused sporulating
cells to generate spore-like structures lacking nuclei
(Fig. 6Bii-iv). This indicates
that Spo3 and Meu14 are both required for the accurate enclosure of the spore
nucleus by the forespore membrane. However, unlike Spo3, whose localization is
distinct from that of Meu14 and stays in the membrane until forespore membrane
development is completed, the intensity of the Meu14-GFP signal at the leading
edge of the forespore membrane diminishes as the forespore membrane grows,
indicating that Meu14 serves a different function to Spo3.
With regard to Don1, its subcellular localization is almost identical to
that of Meu14 except that Don1 appears in the cytoplasm during meiosis I
(Moreno-Borchart et al.,
2001). In contrast, Meu14 appears in the cytoplasm for the first
time at meiosis II (Fig. 5A).
Furthermore, there is no homology in the amino acid sequences of Don1 and
Meu14 and the DON1 gene is not essential for forespore membrane
formation (Moreno-Borchart et al.,
2001
). Thus, Meu14 is distinct from Don1 in both its structure and
function. Taken together, it appears that one function of Meu14 is to guide
the formation of the forespore membrane at its front edge, acting to all
intents and purposes as a size-adjustable steel ring that is used to make a
large soap bubble.
![]() |
Acknowledgments |
---|
![]() |
Footnotes |
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* These authors contributed equally to this work
![]() |
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