1 Division of Biology, Department of Life Sciences, Graduate Program of Arts and
Sciences, University of Tokyo, Tokyo 153-8902, Japan
2 Department of Biological Sciences, Graduate School of Science, University of
Tokyo, Tokyo 113-0033, Japan
3 Department of Cell Biology, National Institute for Basic Biology, Okazaki
444-8585, Japan
* Author for correspondence (e-mail: cnakano{at}bio.c.u-tokyo.ac.jp)
Accepted 27 August 2002
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Summary |
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Key words: Actin cytoskeleton, Cell polarity, Cytokinesis, Microtubule, Rho
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Introduction |
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Molecular genetics is powerful for analyses of cell polarity. The budding
yeast Saccharomyces cerevisiae and the fission yeast
Schizosaccharomyces pombe are generally used as excellent systems for
this purpose (Bähler and Peter,
2000). Budding yeast cells grow asymmetrically by localized
vesicle fusion and cell wall synthesis to form a bud
(Pruyne and Bretscher, 2000a
;
Pruyne and Bretscher, 2000b
).
The bud site assembling-proteins function in assembling components required
for bud growth. During bud formation, patches of F-actin are localized at the
cell surface of the bud, and cables of F-actin are oriented along the axis of
bud formation. In contrast to F-actin, MTs are not required for the bud
emergence and bud growth. Cells of fission yeast are cylindrical in shape and
grow at the ends (Nasim et al.,
1989
). They divide at the mid-region of the cell by contraction of
the contractile ring like many animal cells. In this organism, both F-actin
and MTs contribute to the polarized cell growth again like many animal cells.
Actin is organized as longitudinal F-actin cables and cortical F-actin patches
at the growing ends of the interphase cell where the cell wall is newly
synthesized (Marks and Hyams,
1985
). MTs extend along the long axis of the cell during
interphase (Hagan and Hyams,
1988
) and are essential for maintaining the linear growth of the
cell (Hiraoka et al., 1984
;
Mata and Nurse, 1997
). In
addition, various morphological mutants have been isolated in fission yeast,
which show a round, swollen, bent, or branched shape
(Snell and Nurse, 1993
;
Verde et al., 1995
;
Mata and Nurse, 1998
).
Abnormal organization of F-actin and/or MTs has frequently been observed in
these mutants.
Both the yeasts have two conserved essential Rho family small GTPases, Rho1
and Cdc42, which are counterparts of human RhoA and Cdc42Hs, respectively.
Cdc42 plays an essential role in the establishment of cell polarity in both
yeast cells (Johnson and Pringle,
1990; Miller and Johnson,
1994
), while Rho1 promotes cell wall synthesis for polarized cell
growth (Drgonova et al., 1996
;
Qadota et al., 1996
; Arellano
et al., 1996; Nakano et al.,
1997
; Nakano et al.,
2001a
). In addition, budding yeast has another Rho family protein,
Rho3p, which is involved in maintenance of cell polarity for bud growth
(Matsui and Toh-e, 1992a
). It
has been suggested that budding yeast Rho3p plays this important role by
properly organizing actin cytoskeleton during bud formation
(Imai et al., 1996
). Thus, it
has been desirable to identify and characterize the counterpart of Rho3p in
fission yeast in order to clarify the mechanism of polarized growth of the
cell in general.
Diaphanous/formin family proteins have recently been highlighted since they
seem to play an important role in organizing the contractile ring during
cytokinesis in various cells. Drosophila diaphanous
(Castrillon and Wasserman,
1994), S. pombe Cdc12
(Chang et al., 1997
), S.
cerevisiae Bni 1p (Kohno et al.,
1996
), Aspergillus nidulans SepA
(Harris et al., 1997
), and
Caenorhabditis elegans Cyk1 (Swan
et al., 1998
) are proteins responsible for the cytokinesis in
these organisms. mDia has been shown to localize to the cleavage furrow of
dividing Swiss 3T3 cells (Watanabe et al.,
1997
). It must be noted that some of these proteins has been
considered to be controlled by Rho family proteins: mDia and Bni 1p bind
active forms of RhoA and Rho 1p, respectively. S. pombe has another
diaphanous/formin protein Fus1 which is essential for the mating conjugation
(Petersen et al., 1995
;
Petersen et al., 1998
). Fus1
is expressed when the cells are nitrogen-starved and is localized to the tip
of the mating projection. This formin protein does not appear to function in
polarized growth of the vegetative cell.
Here, we identify Rho3 and demonstrate that it interacts with diaphanous/formin family protein For3 in fission yeast. Analyses of mutants and overexpression phenotypes show that these proteins function cooperatively in regulation of both actin and MT cytoskeletons in polarized cell growth.
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Materials and Methods |
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Isolation of fission yeast rho3+
The budding yeast rho3 null cells (YMR504), which had
been rescued by the expression of the budding yeast RHO4 under the
GAL7 promoter (Matsui and Toh-e,
1992b), were transformed with plasmids containing the fission
yeast cDNA under the control of the budding yeast ADC1 promoter, and
spread on glucose plates. After incubation at 30°C, the growing colonies
were restreaked on glucose plates. Plasmids were recovered from the clones
which grew well on the glucose plates, and were introduced into rho3
null cells to test the ability to complement the defect.
DNA manipulation and cloning of genomic
rho3+
Standard methods were used for the DNA manipulations
(Sambrook et al., 1989). The
procedures for the isolation of rho3+ genomic DNA clones
by plaque hybridization using rho3+ cDNA as a probe were
previously described (Nakano and Mabuchi,
1995
; Nakano et al.,
1997
).
Gene disruption
To disrupt rho3+, we replaced the
SnaI-EcoRV region in EcoRI 3.6 kb genomic fragment
containing rho3+ with ura4+. A
fragment produced by digestion with HincII was used to transform a
diploid constructed by mating JY741 and JY746 strains.
Correct integration was verified by Southern blotting.
To disrupt for3+, we replaced the
SpeI-HpaI region in SmaI-PstI 6.2 kb
fragment containing for3+ with ura4+.
A fragment produced by digestion with SmaI and SacI was used
for gene disruption as mentioned above. for3FH12C,
for3
FH2C, and for3
C were constructed by
inserting ura4+ at 1463, 2589, and 3572 bp in the open
reading frame of for3+, respectively.
Gene expression in fission yeast
S. pombe expression vectors pREP1, pREP41 or pREP81
(Forsburg, 1993;
Maundrell, 1993
) were used.
pREP1 has the strongest promoter, while pREP81 the weakest. The expression of
exogenous genes from these plasmids was repressed by 4 µM thiamine in the
medium.
Two-hybrid system
We used the two-hybrid system in which expression of the HIS3
reporter gene was utilized to detect a positive interaction between two
proteins as described previously (Vojtek
et al., 1993). pBTMrho1Q64L, pBTMrho3, pBTMrho3Q71L, or
pBTMcdc42Q61L contained cDNA encoding the full length of each protein, in
which the Cys in the CAAX-motif was mutated to Ser in order to prevent
isoprenylation of the C-terminus. pGADfor3N contained amino acid residues
149-488 of For3.
Electrophoresis, transfer onto PVDF membranes and western
blotting
Soluble and insoluble protein fractions were prepared from exponentially
growing cultures, subjected to SDS-PAGE, and transfered onto PVDF membranes as
descrived previously (Nakano et al.,
1997). For western blotting, the PVDF membrane was first reacted
with anti-budding yeast Rho3p antibody
(Imai et al., 1996
) and then
Rho3 was detected with secondary antibodies coupled to alkaline phosphatase
(Santa Cruz Co.).
Microscopy
Cells were fixed and processed for immunofluorescence microscopy as
described previously (Alfa et al.,
1993) using anti-budding yeast Rho3 antibodies, anti-HA antibodies
(Y-11; Amersham Pharmacia Biotech), or a monoclonal anti-tubulin antibody
(TAT1; a kind gift from K. Gull). Staining of the cells with Calcofluor,
Rhodamine-phalloidin, or Bodipy-phallacidin (Molecular Probes, Eugene, OR) was
performed as described previously (Alfa et
al., 1993
; Arai et al., 1998). The stained cells were viewed by
two approaches. Conventional images were obtained using a Zeiss Axioskop
fluorescence microscope (Carl Zeiss, Oberkochen, Jena, Germany) equipped with
a Plan Apochromat x63 lens and photographed on Kodak T-MAX ASA 400
films. Three-dimensional (3D) reconstitution was performed using a Delta
Vision system (Applied Precision, Issaquah, WA) attached to an Olympus
IX-70-SIF fluorescence microscope equipped with a UplanApo x100 lens
(Olympus, Tokyo, Japan). The images were captured and processed as described
previously (Motegi et al.,
2001
).
Electron microscopy
Cells were pre-fixed with 2.5% glutaraldehyde dissolved in 0.1 M sodium
phosphate buffer (pH 7.0) at room temperature for 1 hr and then post-fixed
with 1.5% potassium permanganate dissolved in distilled-deionized water at
4°C overnight. They were embedded, thin-sectioned, and examined as
described previously (Nakano et al.,
1997).
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Results |
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rho3 null cells showed defects in cell morphology and
septation
rho3 null cells were normal in both the growth and the shape of
the cells at 25°C (Fig. 1).
However, they grew poorly and displayed aberrant shapes at 37°C; majority
of the cells were dumpy while some were elongated and multi-septated. We also
found that these defects in growth and shape of the rho3 null cells
were suppressed by addition of 1M sorbitol in the medium
(Fig. 1A and data not shown).
Furthermore, almost all the rho3 null cells were multi-septated at
34°C but were still able to grow. These results suggest that Rho3 may be
involved in controlling cell shape and septation. These defects in the
rho3 null cells were suppressed by expression of the budding yeast
RHO3 (data not shown), indicating that the function of Rho3p is
conserved between these organisms. In contrast, a high dose of the fission
yeast Rho1, Rho2, Rho4, or Cdc42 did not suppress the defects of the
rho3 null cells (data not shown). Thus the function of Rho3 in
fission yeast is probably different from that of these Rho family
proteins.
|
Next, we examined distribution of F-actin and MTs in the rho3 null cells (Fig. 1C). At 25°C there was no difference in F-actin- or MT-staining between the rho3 null cells and the wild-type cells. However, at 37°C F-actin patches were delocalized all over the cell cortex and the cytoplasmic MTs were abnormally oriented. In addition, binucleate rho3 null cells without an F-actin ring were occasionally observed, suggesting that cytokinesis was defective in these cells.
Rho3 localizes to the cell periphery
The fission yeast Rho3 was specifically recognized by antibodies raised
against the budding yeast Rho3p (Imai et
al., 1996) (Fig.
2A). Almost all the Rho3 was detected in the 10,000 g
pellet of the cell homogenate. Thus, Rho3 may exist predominantly as a
membrane-bound form like other Rho family small GTPases in fission yeast
(Nakano et al., 1997
;
Hirata et al., 1998
).
|
Next, we investigated cellular localization of Rho3 by immunofluorescence microscopy. Localization of Rho3 in the wild-type cells was very similar to that in the rho3 null cells containing pREP81Rho3 (see below). However, the fluorescence signal in the wild-type cells was too weak to be processed for 3-d imaging (data not shown). Thus, we showed images of the latter cells in Fig. 2B. Rho3 was localized to the cell periphery and relatively concentrated around the growing ends of interphase cells and the mid-region of mitotic cells. The Rho3 staining overlaped with that of F-actin at the very tips of the cell and the septum, but the staining patterns were different: the Rho3 staining was continuous, while F-actin was present as patches. Accumulation of Rho3 at the middle cortex of the cell began at metaphase, progressed during anaphase, and persisted at the septum even during cytokinesis when the F-actin ring contracted. This suggests that Rho3 is not a component of the F-actin ring.
Overexpression of constitutively active Rho3Q71L
Overexpression of Rho3Q71L in the wild-type cells partially inhibited cell
growth and produced both abnormal dumpy cells and mini-cells after removal of
thiamine (Fig. 3). This effect
seemed to be caused by high activity of Rho3Q71L, since both cell growth and
cell shape were not affected by overexpression of wild-type Rho3 (data not
shown). The mini-cells frequently had no nucleus
(Table 2) indicating that cell
division sometimes occurred at an eccentric position of the cell. Staining of
F-actin in mitotic cells showed that formation of the F-actin ring was
incomplete although accumulation of F-actin took place, and that the position
of the F-actin accumulation was often not at the middle of the cell. Electron
microscopy showed that the Rho3Q71L-overexpressing cells formed aberrant
septum: they were often incomplete, not symmetric with respect to the long
axis of the cell, and formed at an eccentric position. Thus, Rho3 is likely to
be involved in controlling both cell shape and septation in fission yeast.
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For3 binds to Rho3
A novel gene encoding the diaphanous/formin family protein
for3+ has recently been reported
(Feierbach and Chang, 2001).
As activated Rho binds to an N-terminal region of some diaphanous/formin
family proteins (reviewed by Wasserman,
1998
), we examined whether any of the Rho family proteins bind to
the N-terminal region of For3 (For3N, amino acid residues 149-488) using the
two-hybrid system (Fig. 4A). A
positive two-hybrid interaction was detected between For3N and Rho3Q71L or
constitutively active Cdc42Q61L, but not between For3N and constitutively
active Rho1Q64L. Wild-types of both Rho3 and Cdc42 also did not show binding
to For3N (Fig. 4A and data not
shown). Thus, it is possible that For3 functions as a target protein for both
Rho3 and Cdc42.
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Localization of For3
We observed cellular localization of For3 by tagging it with an HA epitope
(Fig. 4B,C). HA-For3 was
localized as dots to the ends of the interphase cells and to the middle of
dividing cells. Only a few For3 dots colocalized with F-actin patches at the
cell ends. The For3 dots did not colocalize with F-actin cables. During early
mitosis, the For3 dots seemed to encircle the middle of the cells as a single
ring. However, they were gradually separated from the F-actin ring as double
rings along the septum as cytokinesis proceeded
(Fig. 4B,C). When the cells
were treated with Latrunculin A (Lat-A) in order to disrupt F-actin structures
(Spector et al., 1983), the
For3 dots were partially delocalized (Fig.
4B). Thus, F-actin is likely to be involved in establishment of
the polarized localization of the For3 dots. In addition, For3 did not seem to
colocalize with cytoplasmic MTs as well throughout the cell cycle
(Fig. 4C). Moreover, the
localization of For3 was maintained after disrupting MTs by treatment with
methyl benzimidazole carbamate (MBC) (De
Brabander et al., 1976
) (Fig.
4C). Thus, MTs may not play an important role in the localization
of For3.
Next, we asked whether Rho3 or Cdc42 functions in the localization of For3. First, For3 was localized properly in the rho3 null cells (data not shown). Then, we examined localization of HA-For3 in the rho3 null cells overexpressing wild-type Cdc42 or dominant negative Cdc42T17N, respectively (Fig. 4D). Although F-actin patches were delocalized in the rho3 null cells overexpressing Cdc42, HA-For3 was localized as dots to the ends of interphase cells and to the middle of dividing cells. On the other hand, overexpression of Cdc42T17N caused rounding up of the cell, delocalization of cortical F-actin patches and disappearance of the HA-For3 dots (Fig. 4D).
Furthermore, we investigated a region of For3 molecule, which is required for its localization by expressing For3 and For3-truncated proteins both fused to YFP (an yellow-green variant of the Aequorea victoria green fluorescent protein) (Fig. 4E,F). YFP-For3, YFP-For3NM, YFP-For3N1, or YFP-For3N2 showed a localization pattern similar to HA-For3. In contrast, YFP-For3MC diffused in cytoplasm throughout the cell cycle. Thus, the N-terminal region of For3 is essential for the cellular localization, but other regions are not.
Overexpression of For3 induced formation of thick MT bundles
We examined effect of overexpression of For3 or its truncated proteins in
the wild-type cells (Fig. 5A).
Inhibition of cell growth was seen when For3, For3dC, or For3NM was expressed.
On the other hand, the truncated clone lacking the N-terminal region or the
FH1 domain did not interfere with the cell growth. In addition, overexpression
of For3 inhibited the growth of the rho3 null cells as well as that
of the wild-type cells, suggesting that this inhibition occurred independently
of Rho3.
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Calcofluor staining showed that For3-overexpressing cells formed a bulge in the middle of the cell, and then the cells became swollen (Fig. 5B): 7.3% of these cells displayed the bulge 15 hours after removal of thiamine, while after 20 hours 23% were bulged and 28% were swollen. Moreover, the overexpression of For3 seemed to interfere with septation: 21% of the cells were binucleate without septation at 20 hours after the thiamine removal, while such cells were only 1.1% in the presence of thiamine.
We examined organization of F-actin and MTs in the For3-overexpressing cells (Fig. 5C). F-actin patches were scattered all over the cell cortex. Cytoplasmic MTs formed long bundles which did not end at the cell tips, but lined the cell membrane at the tips and further elongated to the other side of the cell, in contrast to cells not overexpressing For3. Thus, For3 may be involved in controlling organization of both F-actin and MTs in fission yeast.
For3 is involved in controlling cell morphology in fission yeast
for3 null cells were viable
(Fig. 6A), and were slightly
dumpy or bent (Fig. 6B,
Table 3). Cytoplasmic MTs were
often abnormally organized in interphase cells
(Fig. 6C): they tended to be
disordered in the dumpy cells, or curved and located on a convex side of the
bent cells. F-actin patches were moderately randomized. It has very recently
been reported that interphase for3 null cells lack F-actin cables
(Feierbach and Chang, 2001).
However, we were able to find F-actin cables in the interphase for3
null cells although they were reduced in number and short in length as
compared with those in the wild-type cells
(Fig. 6D). In mitotic cells
especially during metaphase, F-actin cables were frequently seen
(Fig. 6C). F-actin ring was
organized normally in the middle of anaphase cells
(Fig. 6C).
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|
We have recently reported that overexpression of an actinbinding domain of
fission yeast fimbrin Fim1 induces formation of thick F-actin cables by
stabilizing pre-existent F-actin cables
(Nakano et al., 2001b). We
thus compared effect of overexpression of this domain (Fim1 A2) in the
for3 null cells to that in wild-type cells
(Fig. 6E). It was found that
thick F-actin cables were formed by overexpressing Fim1 A2 even in the absence
of For3. However, these cables were not straight but curly and often entangled
with each other. For3 may play a role in formation of straight F-actin cables
in interphase cells. Moreover, we investigated a genetic interaction between
For3 and Cdc8 (tropomyosin) since Cdc8 is essential for formation of F-actin
cables probably by stabilizing F-actin (Arai et al., 1998). However, no
synthetic effect was seen in for3 cdc8 double mutant cells (data not
shown). Furthermore, overexpression of Cdc8 did not suppress the defect of the
organization of the F-actin cables in for3 null cells. Thus, For3 may
control the organization of the F-actin cables in a way different from that of
Cdc8.
Next, we investigated a domain of For3 molecule which is involved in
controlling organization of F-actin and MTs. for3+ gene in
for3 FH12C, for3
FH2C, or for3
C strain has an insertion of ura4+ so that
expression of downstream region from amino acid residues 489, 864, or 1192,
respectively, is disrupted. Moreover, we transformed for3 null cells
with pREP1 containing various for3-truncates listed in
Fig. 4E. Expression of a full
length of For3 suppressed the defect of the for3 null cells
(Fig. 4B,C). In contrast, none
of the cells expressing For3-truncated proteins displayed normal organization
of actin cytoskeleton and cytoplasmic MTs
(Fig. 6F and data not shown).
Therefore, each region of For3 is likely to be necessary for its function.
Interestingly, it was found that many interphase F-actin cables were oriented
at random direction in the for3 null cells containing pREP1 For3MC
(Fig. 6Fb). This result is
unlikely to be caused by extraexpression of For3MC since overexpression of
For3MC in wild-type cells does not significantly affect organization of actin
cytoskeleton (data not shown). Thus, it is suggested that For3 lacking the
N-terminal region retains the ability to induce formation of F-actin cables.
Moreover, expression of neither For3FH12 nor For3C induced formation of
F-actin cables in the for3 null cells
(Fig. 6F). Therefore, both the
FH1 domain and the C-terminus of For3 are likely to play important role in
formation of the F-actin cables.
We examined a genetic interaction between Rho3 and For3 by making double null cells. The rho3 for3 double null cells showed severe growth and morphological defects as compared with each single null cells: majority of the cells were swollen including almost round cells (Table 3, Fig. 6G). The double null cells displayed abnormal organization of F-actin and MTs: F-actin patches were delocalized all over the cell surface, and the cytoplasmic MTs were curved and sometimes formed a basket-like structure especially in the round cells. Thus, the defect caused by the depletion of For3 was enhanced in the absence of Rho3.
Next, we examined whether or not depletion of For3 suppresses the effect of overexpression of Rho3Q71L. It was found that the overexpression of Rho3Q71L in for3 null cells caused a defect in the organization of the F-actin ring similar to that in the wild-type cells. Thus, Rho3Q71L can also control actin organization through another pathway which does not include For3. Furthermore, we examined whether the defects caused by the depletion of Rho3 or For3 were suppressed by an increased expression of For3 or Rho3, respectively. No suppressing effect was seen in either case. In addition, defects in organization of both actin cytoskeleton and MTs in for3 null cells were not suppressed by overexpression of Cdc42.
Lastly, we tested whether or not For3 shares function with other S.
pombe diaphanous/formins. It has previously been reported that Cdc12
functions in the assembly of the F-actin ring and both genetically and
physically interacts with Cdc3 (profilin)
(Chang et al., 1997). We thus
examined the phenotypes of for3 cdc12-112 and for3 cdc3-124
double mutants. No synthetic effect was found between For3 and either of these
proteins (data not shown). Furthermore, the for3 null cells were able
to conjugate and sporulate under nitrogen starvation (data not shown),
indicating that For3 is not essential in the process of cell conjugation.
Since fus1+ is essential for the conjugation
(Petersen et al., 1995
), it is
unlikely that For3 exert the function identical to that of Fus1. Thus, the
functions of the three diaphanous/formin family proteins, namely Cdc12, Fus1,
and For3, are probably different from each other in fission yeast.
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Discussion |
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For3 binds to active forms of both Rho3 and Cdc42 but not to that of Rho1.
Cdc42 has been reported to play an important role in establishing cell
polarity: cdc42 deficient cells completely lose the polarity and show
delocalized F-actin patches (Miller and
Johnson, 1994) and basket-like cytoplasmic MTs (K.N. and I.M.,
unpublished). for3 null cells were a little dumpy and also showed
delocalization of F-actin patches and abnormal organization of cytoplasmic
MTs. Therefore, For3 is likely to function through interaction with Rho3
and/or Cdc42 in the control of polarized growth of the cell. Since the
rho3 for3 double null cells showed defects severer than those seen in
each of the single null cells such that round cells appeared, both Rho3 and
For3 play important roles in the polarized growth in the fission yeast
cells.
Increased expression of For3 did not suppress the defect of the
rho3 null cells. This suggests that Rho3 may have another target
protein(s) which functions in a cooperative manner with For3. In budding
yeast, Rho3p directly interacts with type V myosin Myo2p
(Robinson et al., 1999). As
the fission yeast type V myosin, Myo4/Myo52, plays an important role in the
polarized cell growth (Motegi et al.,
2001
; Win et al.,
2001
), it is possible that Rho3 may function in the cell
morphogenesis through both For3 and Myo4. In addition, the budding yeast Rho3p
genetically interacts with Bem1p, Boi1p and Boi2p
(Matsui and Toh-e, 1992b
;
Bender et al., 1996
;
Matsui et al., 1996
). Bem1p
directly interacts with Boi1p, Boi2p, Cdc24p
(Peterson et al., 1994
) and
Ste20p protein kinase (Leeuw et al.,
1995
). These proteins function in the polarized growth of the
budding yeast cell. Recently, it has been reported that Scd1/Ral1, Scd2/Ral3,
Shk1/Pak1/Orb2, and Pob1, which are homologous to budding yeast Cdc24p, Bem1p,
Ste20p, and Boi1p/Boi2p, respectively, play important roles in polarized cell
growth in fission yeast (Chang et al.,
1994
; Marcus et al.,
1995
; Ottilie et al.,
1995
; Sawin et al.,
1998
; Toya et al.,
1999
). Thus, it may also be possible that Rho3 functions
cooperatively with these proteins in addition to For3.
Localization of For3
For3 was localized as dots at the ends of interphase cells which remained
in the absence of either F-actin patches or cytoplasmic MTs. Thus, the For3
localization in the cell cortex is independent of both actin cytoskeleton and
cytoplasmic MTs. Since active forms of both Rho3 and Cdc42 bind For3 and these
proteins localize to the cytoplasmic membrane at the ends of interphase cells
[present study (Merla and Johnson,
2000)], it is likely that For3 associates with these Rho GTPases
during the polarized growth. This is consistent with the fact that the
N-terminal region of For3, including the Rho-binding region, was essential and
sufficient for its cellular localization. Actually, For3 was delocalized in
rho3 null cells overexpressing dominant negative Cdc42T17N. The
N-terminal region of For3 is also required for its function (see below).
Therefore, cortical localization of For3 may be important for its cellular
function.
For3 is involved in organization of both actin cytoskeleton and
microtubules
Both the gene disruption and the overexpression experiments strongly
suggested that For3 may control organization of both F-actin and cytoplasmic
MTs. It was demonstrated that the N-terminal half of For3 including both the
Rho-binding region and the FH1 domain was required for manifestation of the
overexpression effect. The FH1 domain of the diaphanous/formin proteins is
considered to control the organization of the actin cytoskeleton by
interacting with the G-actin-binding protein, profilin
(Wasserman, 1998). Indeed,
fission yeast Cdc12 has been suggested to function in assembling the F-actin
ring by interacting with Cdc3 (Chang et
al., 1997
). However, we were not able to detect any interaction
between For3 and Cdc3. Interestingly, it has very recently been reported that
the budding yeast diaphanous/formin family protein Bni1p stimulates
polymerization of actin in vitro (Pruyne
et al., 2002
; Sagot et al.,
2002a
). Thus, it is possible that For3 controls the actin
cytoskeleton in a direct manner in S. pombe cells.
It is not clear how overexpressed For3 induced the formation of thick MT bundles. The overexpressed For3 was scattered all over the cell cortex as large dots but was not localized to the thick MT bundles (K.N. and I.M., unpublished). Therefore, For3 may influence organization of MTs in an indirect manner. It is possible that For3 in the cell cortex interacts with other proteins which control cytoplasmic MTs. It would be important to identify proteins that associate with the N-terminal half of For3 in order to clarify the mechanisms of regulation of the microtuble cytoskeleton by For3.
We also demonstrated that the C-terminal region of For3 is required for its
function. Both for3 FH2C cells and for3
C cells
were defective in organization of actin cytoskeleton and cytoplasmic MTs like
for3 null cells. Moreover, For3MC exerted an ability to induce
formation of F-actin cables. Interestingly, it has very recently been reported
that the FH2 domain of Bni1p plays an important role in formation of actin
cables in budding yeast (Evangelista et
al., 2002
; Sagot et al.,
2002b
). Moreover, it has also been suggested that mDia in HeLa
cells is possible to control both actin cytoskeleton and MTs through the FH1
and FH2, respectively (Ishizaki et al.,
2001
). Therefore, the FH2 domain of For3 is possible to control
the cytoskeletal organization in the fission yeast. In addition, our results
indicate that the downstream region of the FH2 domain is required for the
function of For3. It has been reported that the C-terminal region of Bni1p
binds to Bud6p/Aip3p (Evangelista et al.,
1997
). A protein homologous to Bud6p/Aip3p has been identified and
revealed to be involved in establishment of cell polarity in S. pombe
(Glynn et al., 2001
;
Jin and Amberg, 2001
).
Therefore, it is possible that For3 interacts with Bud6/Aip3 for controlling
the cytoskeletal organization.
Rho3 is also involved in cytokinesis
Rho3 was localized at the division site during mitosis and septation. The
rho3 null cells formed multi-septated cells above 34°C and some
of the rho3 null cells fail to undergo cytokinesis at 37°C. Thus,
Rho3 may be involved in controlling cytokinesis. Interestingly, overexpression
of Rho3Q71L interfered with proper formation of the F-actin ring both in its
shape and positioning. It has been considered that the position of nucleus is
important for determining the division site in fission yeast
(Tran et al., 2001). However,
the nucleus was correctly located in the Rho3Q71L-overexpressing cells.
Therefore, the control of Rho3 activity may be required for positioning the
cell division plane.
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Acknowledgments |
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