1 Instituto de Microbiología Bioquímica and Departamento de
Microbiología y Genética, Consejo Superior de Investigaciones
Científicas (CSIC)/Universidad de Salamanca, 37007 Salamanca,
Spain
2 Department of Biology, Faculty of Science and Engineering, Konan University,
Okamoto 8-9-1, Kobe 658-8501, Japan
* Author for correspondence (e-mail: ribas{at}usal.es)
Accepted 1 August 2002
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Summary |
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Key words: Fission yeast, Cell wall, Glucan, Septation, Polarized growth, 1,3-ß-D-glucan synthase
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Introduction |
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In the fission yeast Schizosaccharomyces pombe, the major cell
wall structural components are two glucose homopolymers, (1,3)ß-D-glucan
with 2-4% (1,6)ß-D-branches and (1,3)-D-glucan with 7%
(1,4)
-D-bonds, constituting 50-54% and 28-32% of total cell wall
polysaccharides, respectively. A third glucan component, a highly branched
(1,6)ß-D-glucan, accounts for about 2% of total cell wall polysaccharides
(Kopecka et al., 1995
;
Manners and Meyer, 1977
).
Chitin is another important structural component in many fungal cell walls.
Its presence in fission yeast remains controversial, but a chitin synthase
gene, chs1+, and the corresponding enzymatic activity, has
recently been described to be important for spore formation
(Arellano et al., 2000
).
Galactomannan is a non-structural cell wall polymer, linked to proteins to
form glycoproteins and representing 9-14% of cell wall polysaccharides
(Manners and Meyer, 1977
).
(1,3)
-D-glucan synthesis has been attributed to the essential gene
ags1+ (=mok1+), described to encode a
putative catalytic subunit of the enzyme
(Hochstenbach et al., 1998
;
Katayama et al., 1999
). There
are four more mok genes, none of them is essential
(Katayama et al., 1999
) and
their role in cell wall biosynthesis has not been clarified yet.
The (1,3)ß-D-glucan is a major contributor to the cell wall framework.
On this matter, it is relevant to emphasize the only new antifungal agents
under clinical trials are the echinocandins
(Tkacz and DiDomenico, 2001),
whose mode of action, apparently similar to other antifungal drugs, the
papulacandins (Castro et al.,
1995
), is not well known although both clearly interfere with
(1,3)ß-D-glucan synthesis.
The enzyme involved in (1,3)ß-D-glucan synthesis in yeast is the
(1,3)ß-D-glucan synthase (GS), which uses UDP-glucose as substrate,
producing a de novo lineal (1,3)ß-D-glucan
(Cabib et al., 1998). The
activity requires at least two components, a catalytic and a regulatory
subunit (Kang and Cabib, 1986
;
Ribas et al., 1991
). The
latter is the Rho1p GTPase, which drives the activation of GS by GTP
(Arellano et al., 1996
;
Drgonová et al., 1996
;
Qadota et al., 1996
). This
GTPase plays a fundamental role in many morphogenetic processes, particularly
in organization of actin cytoskeleton and genesis of active growing regions
(Arellano et al., 1999
;
Cabib et al., 1998
;
Cabib et al., 2001
).
In budding yeast, two highly homologous genes, FKS1 and
FKS2, encode two putative GS catalytic subunits, which are
differentially expressed depending on the cycle or growth conditions
(Inoue et al., 1995;
Mazur et al., 1995
;
Zhao et al., 1998
). Single
disruption of either FKS1 or FKS2 is viable, but the double
disruption is lethal (Mazur et al.,
1995
). A third FKS gene, found in the Saccharomyces
cerevisiae genome, appears to be nonfunctional.
In S. pombe four FKS homologues have been found. The
first identified gene coding for a putative GS catalytic subunit was cloned by
complementation of the cps1-12 mutant, hypersensitive to a spindle
poison, and named cps1+
(Ishiguro et al., 1997). Later
on, it was also isolated by complementation of mutants defective in septum
formation (Le Goff et al.,
1999b
; Liu et al.,
1999
) and implicated in the coordination between cytokinesis and
cell cycle, being part of a Wee1p-dependent septation checkpoint
(Le Goff et al., 1999b
;
Liu et al., 2000b
). During the
S. pombe genome sequencing project, three other open reading frames
(ORF), whose predicted amino acid sequence share high identity (50-60%) with
Cps1p, have appeared. As other genes already were designated cps
(Ishiguro and Uhara, 1992
),
the cps1+ homologues were named in a common agreement as
bgs (for beta 1,3-glucan synthesis), and therefore
cps1+ has been renamed bgs1+.
bgs2+ is essential for the sexual phase of the life cycle.
Its expression is induced during sporulation and GS activity is diminished in
sporulating bgs2
/bgs2
diploids
(Martin et al., 2000
). Bgs2p
localizes to the ascospore periphery and is required for correct spore wall
maturation and survival (Liu et al.,
2000a
; Martin et al.,
2000
). Finally, to date nothing is known about the role of the
other two homologue genes, bgs3+ and
bgs4+.
While in Saccharomyces cerevisiae there are only two redundant FKS
activities for (1,3)ß-D-glucan synthesis, it is very interesting to
notice that S. pombe contains four different Bgs proteins (and
perhaps the corresponding GS isozymes), to apparently synthesize the same cell
wall component. What is the function and regulation of all of these proteins?
Previous results have pointed to the idea that Bgs1p plays a role in septum
assembly (Ishiguro et al.,
1997; Le Goff et al.,
1999b
; Liu et al.,
1999
). Fission yeast cytokinesis starts with medial ring formation
and contraction. As the medial ring grows centripetally, an intense Calcofluor
white-stained material is concomitantly extruded to the lumen of the
invaginated plasma membrane. Eventually, a disk can be visualized between the
membranes of both daughter cells, which is called, by analogy to the budding
yeast system, the primary septum (Schmidt
et al., 2002
). Subsequently, new cell wall material, the so-called
secondary septum, is laid down at both sides of the primary septum, giving
rise to a complete three-layered septum structure
(Johnson et al., 1973
). In
addition, a recent study describes the in situ localization of different
ß-D-glucans in S. pombe cell wall by using colloidal-gold
labeled specific antibodies (Humbel et
al., 2001
). (1,6)ß-D-glucan-branched (1,3)ß-D-glucan is
localized forming filamentous structures all over the cell wall non-dense
layer and in all the septum. (1,6)ß-D-glucan appears in the same cell
wall non-dense layer, but always close to the outer galactomannan layer, and
in the secondary septum. A linear (1,3)ß-D-glucan is visible only at the
primary septum, as detected by a monoclonal antibody that also specifically
detects the linear (1,3)ß-D-glucan (callose) that constitutes the cell
plate in plant cells (Hong et al.,
2001
).
In this paper, we show genetic and epistatic interactions found between
cps1-12 mutant and several cdc septation mutants, suggesting
that Bgs1p (Cps1p) is involved in the septum cell-wall synthesis process. In
an attempt to elucidate further the Bgs1p function, we have studied Bgs1p
localization during the S. pombe life cycle. In contrast to a recent
report (Liu et al., 2002), in
which it is stated that Bgs1p localizes exclusively to the cell division site,
we have found that Bgs1p localizes first to the growing poles and moves to the
contractile actomyosin ring during septation, remaining in the septum and
reappearing later on at the poles, before the two daughter cells separate. In
addition, we show that Bgs1p is not only restricted to that role, but it is
also present in the meiotic phase of the life cycle, localizing to the mating
projection, cell-to-cell contact zone of fused cells, neck of zygote, and to
or around prospore and spore. Finally, during spore germination, Bgs1p
localizes first, around the spore and next, to the zone of polarized growth.
All these data suggest that Bgs1p is present and could play a role in every
process of the S. pombe life cycle in which synthesis of
(1,3)ß-D-glucan takes place.
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Materials and Methods |
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Standard complete yeast growth medium (YES), selective media (EMM)
supplemented with the appropriate amino acids
(Alfa et al., 1993) and
sporulation medium (SPA) (Egel,
1984
) have been described. The drugs papulacandin B (Ciba-Geigy,
fused to create Novartis) and echinocandin LY280949 (Lilly) were added to
complete (YES) medium, at concentrations ranging from 1.0 to 15 µg/ml.
General procedures for yeast culture and genetic manipulation were carried out
as described (Alfa et al.,
1993
; Moreno et al.,
1991
).
Escherichia coli strains DH5 and DH10B (Life Technologies)
were used for routine propagation of plasmids as described
(Sambrook et al., 1989
), and
CJ236 (Bio-Rad) was used for site-directed mutagenesis. Bacterial LB, 2xYT and
TB media, supplemented with 100 µg/ml ampicillin or 50 µg/ml kanamycin
when appropriate, and standard transformation methods
(Sambrook et al., 1989
), have
been described.
Plasmids and DNA techniques
pCP1 is the 8.04 kb HindIII-HindIII DNA fragment
containing the bgs1+ (cps1+) gene
sequence cloned into the HindIII site of the S. pombe-E.
coli shuttle vector pAL-KS+ (pBluescript KS+ with
ars1+ and S. cerevisiae LEU2 selection)
(Ishiguro et al., 1997;
Tanaka et al., 2000
).
pJR16 is pCP1 with NotI from the multiple cloning site destroyed
(NotI cut and Klenow filled). pJR19 is pJR16 with the ApaI
site from the multiple cloning site destroyed and with an ApaI site
inserted by site-directed mutagenesis just after the bgs1+
TAA stop codon. pJR20 is pJR19 with a NotI site inserted just before
the ATG start codon of bgs1+. pJR24 is pJR20 cut with
NotI-ApaI to eliminate the bgs1+ ORF and
with the ura4+ gene from pBluescript
SK+-ura4+
(Moreno et al., 2000) cut with
NotI-ApaI inserted. This plasmid was used for PCR
amplification of a bgs1+ disruption cassette, as described
below.
pJR17 is pJR16 with a SpeI site inside the
bgs1+ ORF destroyed by site-directed mutagenesis and
making no change in the amino acid sequence of Bgs1p. pJR18 is pJR17 with a
SpeI-SpeI deletion, which removes the 0.79 kb 3'-end
fragment of the cloned bgs1+ sequence. pJR22 is pJR18 with
a 9 bp insertion containing a NotI site just after and a
SpeI site before the ATG initiation codon of
bgs1+ ORF. pJR23 is pJR18 with a 9 bp insertion containing
a NotI site just before and a SpeI site after the TAA
termination codon of bgs1+ ORF. pJG20 is pJR19 with a
HpaI site inserted before the bgs1+ start codon.
pJG25 is pJR1-81XH (81X version of nmt1+
thiamine-repressible promoter and S. pombe his3+
selection) (Moreno et al.,
2000) cut with NruI-ApaI and with the
HpaI-ApaI bgs1+ ORF sequence from pJG20
inserted. The resulting S. pombe strain bgs1
pJG25
displayed wild-type phenotype even in the presence of thiamine, which
represses the regulatable nmt1+ promoter to a very low
expression level (Moreno et al.,
2000
).
pJR92 (pAL-bgs1+-GFP) is pJR23 with an
ApaI-HindIII DNA fragment obtained from PCR amplification of
the 1.57 kb sequence just 5' to the HindIII site of the
promoter sequence (nucleotides -2229 to -661) of the cloned
bgs1+ fragment of pCP1, and also inserted in frame, just
before the bgs1+ TAA stop codon, a 0.74 kb
NotI-NotI fragment from pKS+-GFP
containing a modified GFP-encoding sequence, GFP2-5, which has the changes
S65T, V163A, 1167T and S175G
(Fernandez-Abalos et al.,
1998). Similarly, pJR93 (pAL-GFP-bgs1+) is
pJR22 with the same ApaI-HindIII DNA fragment as in pJR92
and the 0.74 kb NotI-NotI fragment from
pKS+-GFP inserted in frame after the
bgs1+ ATG start codon. pJR98
(pJK-GFP-bgs1+) is the integrative plasmid pJK148 (S.
pombe leu1+ selection)
(Keeney and Boeke, 1994
) with
the ApaI-XbaI GFP-bgs1+ fragment from
pJR93. pJR108 (pJK-bgs1+-GFP) is pJK148 with the
ApaI-XbaI bgs1+-GFP fragment
from pJR92. pJR98 was used to create stable GFP-bgs1+
strains, expressed at physiological level in a bgs1
background.
Site-directed mutagenesis (Kunkel,
1985) was carried out with the Bio-Rad Muta-Gene kit. DNA
sequencing was with a Sequenase kit (Amersham Pharmacia Biotech). Ligation of
DNA fragments was carried out as described
(Moreno et al., 2000
). Plasmid
DNA was introduced into S. pombe cells by an improved LiAc method
(Gietz et al., 1995
). Other
DNA manipulations were carried out essentially as described
(Sambrook et al., 1989
).
Gene disruption
bgs1+ gene disruption was performed in the
Leu- Ura- His- diploid strain 317. The
disruption cassette was made by PCR amplification of a 3.9 kb DNA fragment
from pJR24, which has completely eliminated the bgs1+
coding sequence and contains 0.55 kb of bgs1+ promoter and
5' sequence, the 1.76 kb ura4+ sequence and 1.6 kb
of bgs1+ terminator and 3' sequence. Correct
deletion of bgs1+ ORF was confirmed by PCR analysis using
a combination of oligonucleotides external and internal to the disruption
fragment, either for the 5' end or the 3' end of both
bgs1+ and ura4+ sequences (data not
shown). Tetrads dissection of the
bgs1+/bgs1 strain showed that only the two
Ura- clones of each tetrad were viable, confirming the essential
function of Bgs1p, at least for spore germination, as described for a partial
bgs1+ disruption strain
(Liu et al., 1999
). Haploid
bgs1
strains were obtained by standard random spore analysis
of a bgs1+/bgs1
diploid strain transformed
with the bgs1+-carrying plasmid pCP1 and selection of
Leu+ (pCP1), Ura+ (bgs1
),
His-, Ade- clones.
Enzyme preparation and (1,3)ß-D-glucan synthase assay
Cell extracts and GS assay were essentially as described previously
(Ishiguro et al., 1997). Early
logarithmic phase cells grown at 28°C were washed with buffer A (50 mM
Tris-HCl pH 7.5, 1 mM EDTA and 1 mM ß-mercaptoethanol), suspended in 100
µl of buffer A and broken with glass beads in a FastPrep FP120 apparatus
(Savant; BIO 101) (a 15 second pulse at a speed of 6.0). Broken material was
diluted with buffer A, the cell debris was removed by low speed centrifugation
and the supernatant was centrifuged at 48,000 g for 30 minutes
at 4°C. The pellet was resuspended in buffer A containing 33% glycerol and
stored at -80°C. Standard GS assay mixture contained 5 mM
UDP-D-[14C]glucose (4x104 cpm/200
moles) and
enzyme (20-40 µg protein) in a total volume of 40 µl. All reactions were
carried out in duplicate and data for each strain were calculated from three
independent cultures.
Sensitivity of cells to cell-wall-digesting enzyme complex
Sensitivity of cells to Novozym 234 (Novo Industries) was measured
essentially as described elsewhere
(Ishiguro et al., 1997). The
logphase cells were washed with 50 mM citrate-phosphate buffer (pH 5.6) and
incubated at 30°C with continuous shaking in the same buffer containing
Novozym 234 (30 µg/ml). The residual absorbance of the cell suspensions was
monitored at 600 nm every hour.
Fluorescence microscopy
For F-actin visualization, cells grown in YES liquid medium were fixed with
4% formaldehyde (EM-grade, Polysciences) and stained with rhodamine-conjugated
phalloidin (R-415, Molecular Probes) as described
(Alfa et al., 1993). For
Calcofluor white fluorescence, early-logarithmic phase cells grown in YES
liquid medium were visualized directly by adding a solution of Calcofluor
white to the sample (50 µg/ml final concentration) and using the
appropriate UV filter. For nuclei and cell wall staining, early-logarithmic
phase cells grown in YES liquid medium were fixed with cold 70% ethanol,
centrifuged, resuspended in PBS containing 10 µg/ml Calcofluor white and 20
µg/ml Hoechst (No. 33258, Sigma), and visualized with the same UV filter.
GFP was directly visualized from early-logarithmic phase cells by using the
appropriate UV filter. Calcofluor white or Hoechst and GFP fluorescence do not
interfere with each other and therefore, they can simultaneously be used to
visualize the same cells. Images were obtained using a Leica HC (Germany)
fluorescence microscope, type 020-523.010. For phase contrast and Calcofluor
white-fluorescence a PL APO 40x/0.75 PH2 objective, a Leica DC 100
digital camera and the Leica DC 100 V2.51 software were used. For GFP,
Calcofluor white and Hoechst fluorescence a PL APO 63x/1.32 OIL PH3
objective, a Sensys (Photometrics) digital camera and the Leica QFISH V2.3a
software were used.
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Results |
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In order to study this possibility further, nine double mutants of
cps1-12 with some of the numerous septation mutations, cdc3-6,
cdc4-8, cdc7-24, cdc8-134, cdc11-136, cdc12-112, cdc14-118, cdc15-140 and
cdc16-116, representative of different septation steps (reviewed by
Gould and Simanis, 1997;
Le Goff et al., 1999a
), were
constructed. Four double mutants, cps1-12 with cdc3-6, cdc14-118,
cdc15-140 and cdc16-116, were much more sensitive to Novozym 234
than the parental single mutants (data not shown). In addition, the former
four double mutants were more thermosensitive, either in the absence or in the
presence of 1.2 M sorbitol, and exhibited abnormal cell morphology and actin
distribution at conditions in which the single mutants showed no alteration
(data not shown). Moreover, a double mutant of cps1-12 with the actin
mutant cps8-188 (Ishiguro and
Kobayashi, 1996
) was found to be more thermosensitive (data not
shown) and to have abnormal cell morphology at conditions in which the single
mutants were essentially normal (Fig.
1). Furthermore, the four cdc and cps8-188
double mutants with cps1-12 presented variable increased
hypersensitivities to the cell wall and membrane disturbing detergent SDS
(Bickle et al., 1998
;
Igual et al., 1996
) and to the
GS inhibitors papulacandin B and echinocandin, and a slightly altered GS
activity (from 70% to 120% wild-type activity, data not shown). All these data
suggest a certain genetic interaction of actin and septation mutants with
cps1-12, which are somehow increasing its cell wall-related
phenotypes and altering its role in GS activity.
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cps1-12 single and double mutants were also analyzed for their
morphology phenotypes at different temperatures. The cells were visualized by
phase-contrast and by Calcofluor white UV staining, which preferentially binds
to cell wall material, probably the (1,3)ß-D-glucan, of septum and
growing zones. At the corresponding temperature, an epistatic interaction
between cps1-12 and these cdc mutants
(Fig. 1) was found. Three of
the mutants, cdc3-6, cdc14-118 and cdc15-140, are involved
in steps prior to the beginning of septum synthesis
(Gould and Simanis, 1997;
Le Goff et al., 1999a
). In
these double mutants, the dominant phenotype was that of the cdc
mutant (Fig. 1 and data not
shown). This result is in agreement with that described for double mutants of
other cps1 mutant alleles and some cdc mutants involved in
steps prior to septum synthesis (Le Goff
et al., 1999b
; Liu et al.,
1999
). The last mutant, cdc16-116, is involved in
stopping septum synthesis, and in this case, the dominant double mutant
phenotype was that of cps1-12
(Fig. 1). The same result was
obtained in the presence of 1.2 M sorbitol (data not shown). Therefore, these
results suggest that Bgs1p is involved in the septum synthesis process, acting
in a window between the beginning and end of septum synthesis, in agreement
with the proposed function of Bgs1p as a possible GS catalytic subunit
(Ishiguro, 1998
;
Ishiguro et al., 1997
).
Bgs1p localizes to the septum during cytokinesis and to one or both
poles during cell growth
As the genetic results seem to implicate Bgs1p in the synthesis of septum
cell wall, we were interested in knowing the physiological localization of
Bgs1p along the cell cycle. GFP-Bgs1p, but not Bgs1-GFP, expressed either from
multicopy or integrative plasmids restored completely the wild-type phenotype
to a bgs1 strain and the GFP fluorescence appeared localized
as well. Therefore, a bgs1
strain containing an integrated
GFP-bgs1+ copy (single copy, own promoter and absence of
original bgs1+ gene) was chosen for Bgs1p localization
studies.
Calcofluor white staining shows a marked fluorescence at one pole during
monopolar growth and at both poles when the cell grows in a bipolar fashion.
Somewhat unexpectedly, Bgs1p was found localized not only to the septum but
also to the growing ends along the mitotic cycle, overlapping the Calcofluor
white staining (Fig. 2A).
Initially Bgs1p accumulates at one pole, then it also appears at the opposite
pole and finally, the GFP disappears from both poles and localizes to the
middle of the cell, where it concentrates into a narrow ring. The GFP signal
is strongest where the ring is intersected by the focal plane, and it moves
centripetally with the inner edge of the growing septum. A fainter signal
remains along the invaginated membrane where new Calcofluor white-stained cell
wall material has been made (Fig.
2B). When the cell-wall septum is completed, the GFP appears in
two separated bands. During cell division Bgs1p remains at this pole. Finally,
before the two daughter cells separate and the Calcofluor white staining
disappears, Bgs1p changes its localization to the old end of both cells, which
are then ready to initiate the monopolar growth
(Fig. 2A,B). Our Bgs1p
localization results differ from those recently published by other authors
(Liu et al., 2002), in which
Bgs1p is detected only to the division site. Possible reasons why they find
Bgs1p exclusively localized to the cell division site are given in the
Discussion. In summary, Bgs1p localization correlates with septation and
polarized cell growth, suggesting that Bgs1p is responsible for the synthesis
of both a specific (1,3)ß-D-glucan that constitutes the fission yeast
primary septum and part of the (1,3)ß-D-glucan that is made at the poles
during cell growth.
|
Bgs1p septum localization depends on the medial ring and the
septation initiation network
In order to know whether Bgs1p localization at the middle of the cell is
dependent on the presence and/or localization of the medial ring, several
septation mutants were made in a bgs1
GFP-bgs1+ background and GFP-Bgs1p localization was
analyzed (Fig. 3).
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Mid1p is essential for proper positioning of the medial ring, arriving at
the zone before other ring proteins (Bahler
et al., 1998; Paoletti and
Chang, 2000
; Sohrmann et al.,
1996
). In mid1 mutants, actin ring and septum are
positioned at random locations and angles on the cell surface
(Chang et al., 1996
). The
mid1-366 mutant grown at the restrictive temperature showed that ring
and septum alterations were coincident with Bgs1p localization and with the
Calcofluor white-stained cell wall material
(Fig. 3). Cdc3p, or profilin,
is essential for medial ring assembly
(Balasubramanian et al., 1994
).
The cdc3-6 mutant grown at the restrictive temperature is defective
in septum formation but not in cell elongation
(Nurse et al., 1976
),
accumulating Calcofluor white-stained cell wall material at the site of septum
formation (Ishiguro et al.,
2001
). In this mutant, Bgs1p was detected at the poles and/or
coincident with many of the cell wall structures synthesized at the middle of
the cell (Fig. 3). Cdc15p is
essential for cell division. The mutants have impaired the actin ring
formation or form unstable ring structures, and display disorganized actin
patches during mitosis (Balasubramanian et
al., 1998
; Chang et al.,
1996
; Fankhauser et al.,
1995
). The cdc15-140 mutants have thick or diffuse cell
wall depositions at both sides of the cell, making aberrant structures
resembling the septa but in which Bgs1p was never present. As the mutants may
continue growing, Bgs1p was sometimes present at one cell pole. Part of Bgs1p
also remained in ring or septum structures made during the temperature shift
of the mutant and before expressing the thermosensitive phenotype
(Fig. 3). Cdc11p
(Balasubramanian et al., 1998
;
Krapp et al., 2001
;
Mitchison and Nurse, 1985
;
Nurse et al., 1976
) and Cdc14p
(Fankhauser and Simanis, 1993
;
Guertin et al., 2000
) are
essential for septum formation. The cdc11-119 and cdc14-118
mutations block cytokinesis but assembly of the actomyosin ring or
accumulation of actin patches at late anaphase during mitosis or at the cell
ends during interphase is not affected. In cdc11-119 and
cdc14-118, Bgs1p localized only to the poles, coincident with cell
growth and new cell wall synthesis. Some mutant cells presented
Calcofluor-stained ring or septum structures and cdc14-118 also
showed some diffuse cell wall synthesis at the middle of the cell, in a
septum-like manner, but Bgs1p was never present in any of these structures
(Fig. 3). Cdc16p is required
for negative regulation of septum formation, by forming a two-component GAP
for Spg1p GTPase (Cerutti and Simanis,
1999
; Fankhauser et al.,
1993
; Furge et al.,
1998
). The cdc16-116 mutant fails to turn off septation,
making multiple septa in the cell (Minet
et al., 1979
). Bgs1p appeared at the poles and at many of the
multiple septa (Fig. 3). In
addition, Bgs1p localization in cdc15-140 and cdc16-116 was
affected, increasing the fluorescence background inside the cell and making
Bgs1p localization undetectable at longer incubations at the restrictive
temperature (data not shown). In summary, the contractile ring is necessary
but not sufficient for Bgs1p localization to the middle of the cell. The
proteins that form part of the septation initiation network (SIN), such as
Cdc11p and Cdc14p, are also necessary for its localization.
Next, we wondered whether the SIN proteins are necessary not only for stable Bgs1p localization to the medial ring, but also for Bgs1p displacement from the tips to the medial ring during mitosis. For that purpose, Calcofluor white and nuclei staining and GFP-Bgs1p were simultaneously analyzed in the same cells. In wild-type cells, Bgs1p was localized to both cell ends until the end of anaphase, when nucleus division is completed and the nuclei are separated at both cell poles. Next, Bgs1p disappeared from the poles and two GFP dots appeared at the middle of the cell. Finally, two Calcofluor-stained dots appeared overlapping the bright GFP signals (data not shown). If SIN signaling is not necessary for Bgs1p displacement from the tips, the SIN mutants will show post-anaphase Bgs1p displacement from the tips to the contractile ring and its reappearance at the tips when end growth resumes. Alternatively, if SIN signaling is necessary, Bgs1p will never be detected at the medial ring. Analysis of cdc11-119 and cdc14-118 mutants after 2, 4 and 6 hours of growth at the restrictive temperature, containing two, four and eight nuclei after completion of one, two or three mitotic cycles, showed that Bgs1p never relocated to the medial ring, even in those cells that contained a partial septum structure (Fig. 4). Bgs1p was detected in the medial ring only at the beginning, before expressing the thermosensitive phenotype, and at late times, when the mutants escaped the phenotype and started to make complete septa (data not shown). Therefore, SIN signaling is required for Bgs1p displacement from the tips to the contractile ring.
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The actin cytoskeleton is essential for correct Bgs1p
localization
F-actin localizes to either the growing tips or the medial region of
dividing cells (Marks et al.,
1986), where new cell wall material is being made, and overlaps
Bgs1p localization along the cell cycle. In addition, the cps1-12
mutant showed genetic interactions with the actin mutant cps8-188.
Therefore, we were interested in analyzing whether the actin cytoskeleton is
involved in correct Bgs1p localization. In the cps8-188 mutant grown
at 37°C, the actin remains polymerized but the dots are randomly
distributed throughout the cell and the middle ring structure is absent
(Ishiguro and Kobayashi,
1996
). The cells are rounded and enlarged, and some of them
display a septum but do not divide. As the mutant increased its phenotype of
random actin distribution, Bgs1p localization extended from poles or septum
along the plasma membrane until it was located all around the cell
(Fig. 5). The cells also
presented areas of more intense GFP fluorescence in septum or cell periphery,
coincident with regions of delocalized cell wall synthesis. Besides, the cells
showed a substantial GFP-Bgs1p accumulation inside the cell. The
cps8-188 mutant grown at 32°C displays an intermediate phenotype
of enlarged and aberrant cells that can still form aberrant contractile ring
structures. GFP-Bgs1p was localized as a broad band in cell poles and septum
simultaneously, and in regions of aberrant cell wall accumulation. Bgs1p was
also present in abnormal contractile ring and septum structures, and part was
dispersed inside the cell (Fig.
5).
|
The actin structures were disrupted in the profilin mutant cdc3-6 after 5 hours of growth at 37°C. The cells showed aberrant septa and cell wall structures, but Bgs1p never localized there. Instead, it appeared delocalized in the cytoplasm as a low GFP background (data not shown). In summary, first Bgs1p localization is dependent on the presence of a polymerized actin cytoskeleton, and second the specific Bgs1p localization depends on the correct localization of polymerized actin.
Bgs1p localization is altered in the polarity establishment mutants
tea1-1 and tea2-1
Bgs1p not only localizes to the septum but also to the poles during both
monopolar and bipolar growth. Tea1p and Tea2p are end markers, required to
establish the correct growth site and therefore, they might also be
responsible for Bgs1p localization during polarized growth. For that purpose,
GFP-Bgs1p was analyzed in strains tea1-1, tea2-1 and tea2-1
cdc11-119 (Fig. 6).
tea1-1 and tea2-1 mutants do not recognize the old end after
mitosis, becoming branched by activating a new incorrectly positioned growing
site, and the double mutant tea2-1 cdc11-119 activates several new
growing ends (Browning et al.,
2000; Mata and Nurse,
1997
; Verde et al.,
1995
). In the tea1-1 and tea2-1 mutants, Bgs1p
appeared localized to the incorrect new growing end. During mitosis, the
mutants also showed mislocalization of contractile ring and septum, often
initiating ring contraction from three different points and dividing the cell
into three compartments. In all cases, Bgs1p was polarized, appearing at the
growing tips or in the altered septa (Fig.
6). In the tea2-1 cdc11-119 mutant, Bgs1p localized to
multiple new growing ends (Fig.
6).
|
These results indicate that Bgs1p localization in these mutants remains polarized to the growing poles and septum, which are mislocalized due to the failure of the polarity system to establish and maintain the polarized growth along the long axis of the cell.
Bgs1p is also present and localized to the growing zones during
mating, spore wall formation and spore germination
Bgs1p shows a localized pattern in all stages of vegetative growth. As it
is present at every point where growth takes place, we wondered whether it
might also be present at the cell wall growth sites during the sexual phase of
the life cycle. Thus, Bgs1p localization was evaluated along the sporulation
process in a homothallic bgs1 GFP-bgs1+
h90 strain.
During the mating process, Bgs1p appears well localized to the mating projection of the cells (Fig. 7). Initially, Bgs1p is found in only one of both cells, even before any mating projection is observed. When the mating projection is present, Bgs1p is even more apparent. Then, the other cell begins to respond to the call of the first cell, and Bgs1p and the mating projection appears in the second cell. Then, the cell walls of both cells fuse and Bgs1p stays as a band in the septum that separates both cells. When the cells fuse and the septum disappears, Bgs1p appears as a wide band at both sides of the neck between both cells (Fig. 7); it then disappears first from one side of the neck-membrane and then from the other. At that time, Bgs1p is filling in all the cytoplasm, uniformly and with intense fluorescence, but is absent at the fused nucleus, which extends along the zygote and divides, giving rise to the four nuclei. While remaining in the cytoplasm, Bgs1p begins to appear inside or associated with the membranes of the four well-defined oval or round shaped forespores (Fig. 7). Bgs1p disappears from the cytoplasm, remaining in the prospores and accumulating as very bright patches that will remain during the rest of spore maturation processes. Again, Bgs1p appears in the cytoplasm as uniform and extremely bright fluorescence. During all this process, Calcofluor white only shows the zygote shape, with no staining of prospore cell wall, although the prospores are perfectly distinguishable at phase contrast microscopy (data not shown). At this point, Calcofluor white begins to stain the spores inside the ascus, probably because the prospore is maturating its wall to become a spore (Fig. 7). Next, Bgs1p accumulates outside the spores as bright and random patches, usually close to the spore wall, and then, the general Bgs1p fluorescence all around the cytoplasm gradually disappears, until it only remains localized to the entire spore periphery and as bright patches inside the mature spores. At that time, the spores are completely mature and the envelope that forms the ascus will eventually lyse to release the spores (Fig. 7). All these observations suggest that Bgs1p function extends beyond the vegetative cycle. According to its specific localization pattern, Bgs1p might have a dual role during mating: first, helping the cell to grow and form the mating projection; and second, forming part of a security mechanism that compensates any excess of cell wall degradation during the cell fusion process. In addition, these observations also suggest that Bgs1p has multiple roles during sporulation, helping to synthesize both prospore and spore walls.
|
To analyze whether Bgs1p is also present during the spore germination
process, germinating spores of homothallic bgs1
GFP-bgs1+ h90 strain were examined
(Fig. 8). In order to avoid any
treatment with a cell wall hydrolytic enzyme complex that might alter the
spore wall structure and perhaps modify Bgs1p localization, the spores were
released by spontaneous lysis of the asci during a long incubation period in
SPA solid medium. The spore analysis showed that Bgs1p is also involved in
several germination steps (Fig.
8). Initially, the spores show an isotropic growth. Bgs1p
reorganizes, causing the internal bright patches to disappear and become
distributed uniformly in the spore periphery and weakly inside the spore
(Fig. 8). Then, Calcofluor
white staining shows the beginning of a polarized growth and Bgs1p increases
its localization to that place. The spore elongates by a monopolar growth and
Bgs1p is always present at this growth pole. When the spore stops growing,
Bgs1p reorganizes to the middle of the cell, being first with the contractile
ring and later in the spore septum. During cell division, Bgs1p remains
between the spore and the new cell. Before division is completed, it localizes
to the old end of the new cell, but remains in the septation pole of the
spore, probably because the spore will reinitiate its growth at this pole,
which resembles a vegetative cell.
|
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Discussion |
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---|
In this work, we present a series of genetic and localization results,
which suggest that Bgs1p function is implicated in septum synthesis. We show
that the cps1-12 mutant interacts genetically with some cdc
septation mutants. The double mutants displayed several aggravated phenotypes.
Interestingly, some septation mutants did not show negative interaction with
cps1-12, measured as increased sensitivity to Novozym 234. Perhaps
the former class of septation mutants affects the cell wall integrity in a
different way compared with the cps1-12 mutant, whereas the latter
class might be involved in the Bgs1p pathway itself. However, additional
experiments are needed to support this hypothesis. Another reason why some
septation mutants did not show negative interaction with cps1-12
could be the specificity of the bgs1 mutant allele. In fact, similar
results have been described for other bgs1 mutant alleles and some
cdc septation mutants, including cdc mutants that in our
case show negative interaction and vice versa
(Le Goff et al., 1999b;
Liu et al., 1999
). We also
describe epistatic interactions between cps1-12 and the cdc
septation mutants. Similar epistatic interactions have been described for
other bgs1 mutant alleles (Le
Goff et al., 1999b
; Liu et
al., 1999
) but in these cases all the analyzed septation mutants
were epistatic to the bgs1 mutants and, therefore, Bgs1p function was
not well localized as it could act after septum synthesis. In our case, we
showed that cdc16-116 is epistatic to cps1-12, which
suggested that Bgs1p function is located in the period between the beginning
and end of septum synthesis at cytokinesis. This result is consistent with the
proposed function of Bgs1p as one of the S. pombe GS catalytic
subunits, and in that case, Bgs1p would be responsible for septum cell-wall
(1,3)ß-D-glucan synthesis.
We were interested in studying Bgs1p localization along the cell cycle. For
this purpose, we constructed a bgs1+/bgs1
mutant strain that had the complete bgs1+ ORF removed.
Sporulation of this strain showed bgs1+ is essential at
least for spore germination. A similar result was obtained for a mutant with a
partial disruption of the bgs1+ gene
(Liu et al., 1999
).
Germination of bgs1
spores results in spherical and enlarged
cells that eventually will lyse (Liu et
al., 1999
). This phenotype points to the importance of Bgs1p
localization for polarized cell growth, at least during spore germination.
Although it is likely that bgs1+ is also essential for
vegetative cells, it has not been proven yet. In fact, repression of the
bgs1+ gene by the 81X version of the thiamine-repressible
nmt1+ promoter (pJG25) is not lethal in a
bgs1
strain (see Materials and Methods). Nevertheless, we
cannot discard the possibility of a residual bgs1+
expression, high enough to maintain cell viability.
In order to avoid any artifact in Bgs1p localization studies, we tried to
mimic the physiological state for Bgs1p. An integrated single copy of
GFP-bgs1+ gene, regulated by its own
bgs1+ promoter, made the bgs1 mutant
revert to the wild-type. Therefore, GFP-Bgs1p is functional and expressed at
physiological levels. During septum synthesis, Bgs1p localization is
coincident with that of the contractile ring, appearing as bright fluorescence
signals ahead of the newly synthesized septum cell wall. These results suggest
that Bgs1p might be responsible for the synthesis of a specific lineal
(1,3)ß-D-glucan that constitutes the fission yeast primary septum
(Humbel et al., 2001
) just as
CalS1p is responsible for the formation of the cell plate in plant cells, made
of callose (Verma and Hong,
2001
), in a process that mimics the synthesis of the primary
septum in S. cerevisiae, made of chitin, by Chs2p
(Schmidt et al., 2002
).
Nonetheless, S. cerevisiae might present some differences, as ring
contraction and primary septum formation depend on each other
(Schmidt et al., 2002
).
Initially, our results of Bgs1p localization indicate that its requirements
are coincident with those of S. cerevisiae: Bgs1p localization
depends on the presence of an actomyosin ring and on its contraction, promoted
by the septation initiation network (SIN)
(McCollum and Gould, 2001
;
Sawin, 2000
). However,
cps1-12 mutant may apparently form normal septa when grown under
certain conditions, although the cells are unable to separate. If the
cps1-12 mutant phenotype were due to the absence of Bgs1p function
and if its function were the primary septum synthesis, this would mean that
the medial ring can contract and a septum can be made, even in the absence of
primary septum.
After submitting this manuscript for publication, another work describing
Bgs1p localization appeared (Liu et al.,
2002). The described localization data and conclusions are
coincident with part of the results presented in our work. The authors reach
the same conclusions about Bgs1p localization to the contractile ring and its
dependence on F-actin and SIN proteins for its localization. Furthermore, it
is stated that Bgs1p is not required for cell elongation and is visualized
only in cells undergoing mitosis and cytokinesis. It is not detected in
disc-like structures following assembly of the primary septum. In our case,
Bgs1p is clearly detected in every process of cell wall growth. We do not know
the exact reason why it was not detected by Liu et al., but it is probably due
to one of the following possibilities. First, Bgs1p localization is very
unstable and extremely sensitive to cell wall stress, as has been reported for
the S. cerevisiae homologue Fks1p
(Delley and Hall, 1999
). In
the studies by Liu et al., it is likely that the cells lost Bgs1p from the
poles or septum during manipulation. In our experiments, despite careful
handling of the cells, the GFP sometimes disappeared from the cell poles and
accumulated inside the cell after two or three pictures or even at the
beginning. Moreover, we noticed different degrees of Bgs1p stability: very
unstable at the poles, more stable along the septum and very stable at the
contractile ring. However, after ethanol fixation, Bgs1p localization seemed
to be stable throughout the cell. Second, the GFP-Bgs1p fusion protein used by
Liu et al. might display a different behavior. They did not describe whether
the gene replacement was done in haploid or diploid strains. If the fusion
protein is not completely functional, the gene replacement in haploid strains
might promote gene duplication. In that case, wild-type Bgs1p could be more
stable or have more affinity for growth poles than the fusion protein.
Finally, this specific fusion protein might be less stable, making its
detection outside the contractile ring more difficult.
Bgs1p localization in tea mutants suggests that it depends on the
microtubule-dependent growth establishment. In addition, it has been
demonstrated that actin is another key component directing Bgs1p localization,
as it is for Ags1p, the putative (1,3)-D-glucan synthase
(Katayama et al., 1999
).
Disappearance of Bgs1p fluorescence from the poles at the time of cytokinesis
and vice versa, favors the idea of a mechanism for a drastic and rapid Bgs1p
recruitment to the medial ring zone at the time of septum formation and to the
poles at the time of cell growth, coincident with the location of actin
patches and cytoskeleton components at the same region of active cell-wall
synthesis. In fact, it has recently been shown that a GFP fusion of the S.
cerevisiae homologue Fks1p moves on the cell surface to the sites of cell
wall synthesis. This Fks1p mobility is dependent on the movement of cortical
actin patches and is necessary for correct cell wall synthesis
(Utsugi et al., 2002
).
It is likely that Bgs1p is an integral constituent of the plasma membrane.
Therefore, its recycling or degradation is probably channeled through
endocytosis. Our observation of intermediate stages of cells with Bgs1p
localized between cell poles and septum, and of growing cells with
internalized Bgs1p, revealed the presence of lumpy and granular Bgs1p
structures in the cytoplasm. This suggests that Bgs1p is processed by
endocytosis rather than diffusing freely. If this were the case, it would be
especially interesting to search for the final destination of Bgs1p. This
processing might lead to complete degradation in the vacuole or lysosome, or
to an alternative route of Bgs1p recycling, as has been described for S.
cerevisiae chitin synthases Chs2p and Chs3p, respectively
(Chuang and Schekman,
1996).
The presence of Bgs1p in the growing sites during the mitotic cycle is
coincident with the localization of Rho1p GTPase, the GS regulatory subunit
(Arellano et al., 1997). This
colocalization of both proteins is consistent with the idea that Bgs1p is
probably a GS catalytic subunit or forms part of a GS catalytic complex.
However, it is still not known whether Rho1p has a function and/or localizes
to the same sites during mating, sporulation and spore germination.
All this work suggests that the Bgs1p role is widely extended along the
life cycle, probably involved in the synthesis of all or part of the cell wall
(1,3)ß-D-glucan. Alternatively, it might also have a cell wall repair or
protection function during cell growth or zygote formation. This is consistent
with the idea there are three Bgs1p homologues, which might have specific and
separate functions during cell wall synthesis, as is the case for chitin
synthesis in S. cerevisiae (Shaw
et al., 1991). Supporting this idea, the Bgs2p homologue has been
found to be essential for sporulation but not for vegetative growth
(Liu et al., 2000a
;
Martin et al., 2000
).
Furthermore, bgs3+ and bgs4+ have
already been cloned and both are essential genes (M. V. Martin, B. Garcia, E.
Carnero, A. D. et al., unpublished; J. C. G. C., J. I., A. D. et al.,
unpublished). Moreover, bgs4+ complements all
cwg1-1 mutant phenotypes (Ribas
et al., 1991
), which display reduced GS activity and cell wall
(1,3)ß-D-glucan (J. C. G. C., I. P. Martins, E. Carnero et al.,
unpublished). An appealing idea is that Bgs3p and Bgs4p would be responsible
for other essential (1,3)ß-D-glucans that form the cell wall and
secondary septum, either by synthesizing different types of glucan or by
displaying a differential localization, either spatial or temporal, along the
cell cycle. This would also be the case for Bgs1p during sporulation. Bgs2p
has been found to be essential for sporulation, but it does not imply that all
the (1,3)ß-D-glucan made in the spore wall is due to Bgs2p. In fact, a
further Bgs1p study will be necessary to ascertain its specific role for spore
wall formation.
S. pombe is the only described model system with four different
essential Bgs proteins to apparently synthesize the same (1,3)ß-D-glucan.
What is the function and regulation of any of them? It is believed that they
form part of the GS catalytic subunit but none contains the proposed
UDP-glucose binding consensus R/K-X-G-G of glycogen synthases
(Farkas et al., 1990;
Furukawa et al., 1990
).
Instead, they all contain the proposed consensus RXTG
(Inoue et al., 1996
;
Ishiguro et al., 1997
), but
more experimental work will be needed to prove their function as GS catalytic
subunits as well as to ascertain the specific role of each Bgs subunit in the
fission yeast life cycle.
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Acknowledgments |
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