1 Division of Biology, Department of Life Sciences, Graduate School of Arts and
Sciences, University of Tokyo, Tokyo, 153-8902, Japan
2 Department of Cell Biology, National Institute for Basic Biology, Okazaki,
444-8585, Japan
* Author for correspondence (e-mail: mabuchi{at}ims.u-tokyo.ac.jp )
Accepted 19 November 2001
![]() |
Summary |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: F-actin ring, F-actin cable, Aster-like structure, Deconvolution, 3D reconstruction
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The fission yeast Schizosaccharomyces pombe has three simple
F-actin structures: cortical patches, cables and rings
(Marks and Hyams, 1985).
Dynamic behaviour of these actin cytoskeletons during the cell cycle has been
investigated (Marks and Hyams,
1985
; Arai et al.,
1998
). The F-actin patches localize to both growing ends of the
cylindrical cell during interphase. It is suggested that the F-actin patches
play a role in the deposition of cell wall materials and in the maintenance of
polarized cell growth (Kobori et al.,
1989
; Ishiguro and Kobayashi,
1996
). The localization of F-actin patches has been shown to be
controlled by small GTP-binding proteins and various actin-modulating proteins
(for reviews, see Ishiguro,
1998
; Le Goff et al.,
1999
). The F-actin cables run longitudinally during interphase and
seem to be linked to some F-actin patches. During mitosis, the F-actin patches
disappear from the ends of the cell, and the F-actin ring is formed at the
medial region of the cell. The F-actin cables are seen to attach to the
F-actin ring during mitosis (Arai et al.,
1998
). It has been proposed that F-actin ring formation initiates
before anaphase in S. pombe
(Marks and Hyams, 1985
), being
different from animal cells (Mabuchi,
1994
) or Schizosaccharomyces japonicus
(Alfa and Hyams, 1990
), in
which the contractile ring is formed during late anaphase to telophase.
Recently, it has been reported that the actin filaments first appear as a
faint and broad F-actin ring in the wild-type S. pombe cell before
anaphase. However, how the actin filaments accumulate at the medial region
remains unknown. Then the ring becomes a sharp one as anaphase progresses
(Arai et al., 1998
;
Bähler et
al., 1998
). As the ring shrinks during cytokinesis, septum
invaginates centripetally from the cell surface. The F-actin patches
relocalize near the septum region in this stage.
Several cell division mutants that cannot form the F-actin ring and grow
into large multinucleate cells at the restrictive temperature have been
obtained in S. pombe, and several proteins that are involved in the
F-actin ring formation have been reported and characterized by analyzing these
mutants (for a review see Le Goff et al.,
1999). Some are considered to function in early steps of the ring
formation process. Plo1, a polo kinase homolog, is an important protein to
regulate and initiate the F-actin ring formation. When Plo1 is overexpressed,
F-actin ring formation is induced even in interphase
(Ohkura et al., 1995
).
mid1/dmf1/pos1 mutant cells form F-actin rings and septa at a random
position and angles (Chang et al.,
1996
; Sohrmann et al.,
1996
; Edamatsu and Toyoshima,
1996
). Thus, Mid1 is required to determine the position of the
F-actin ring formation. It localizes on the nuclear membrane during interphase
(Sohrmann et al., 1996
;
Bähler et
al., 1998
), but relocalizes as a broad ring at the medial region
of the cell during prophase, and then the ring becomes sharp
(Bähler et
al., 1998
). Recently, it has been reported that Plo1 is necessary
for the proper Mid1 function
(Bähler et
al., 1998
).
Mutants of the cdc12 and cdc15 genes, which encode a
diaphanous family protein and an SH3-containing protein, respectively, cannot
form the normal F-actin ring during mitosis, but display normally localized
F-actin patches during interphase
(Fankhauser et al., 1995;
Chang et al., 1996
;
Balasubramanian et al., 1998
).
Cdc12 localizes as a spot in the cytoplasm during interphase when it is
overexpressed in the cell (Chang et al.
1997
; Chang, 1999
).
During prophase, the Cdc12 spot positions at the medial cortex. A single Cdc12
strand extends from the spot and then encircles the cell at the equator
(Chang et al., 1997
). However,
when Cdc15 is overexpressed in G2-arrested cells, F-actin ring formation is
induced (Fankhauser et al.,
1995
). These phenotypes suggest that both Cdc12 and Cdc15 play
specific roles, in an early step of F-actin ring assembly. In mutants of
cdc3 and cdc8 genes, which encode profilin and tropomyosin,
respectively, both formation of the F-actin ring and localization of the
F-actin patches are impaired
(Balasubramanian et al., 1992
;
Balasubramanian et al., 1994
;
Chang et al., 1996
).
Tropomyosin is also required in organization of the F-actin cables
(Arai et al., 1998
).
Type II myosin heavy chains, Myo2
(Kitayama et al., 1997;
May et al., 1997
) and
Myp2/Myo3 (Bezanilla et al.,
1997
; Motegi et al.,
1997
), and light chains Cdc4
(McCollum et al., 1995
) and
Rlc1 (Le Goff et al., 2000
;
Naqvi et al., 2000
) are
localized only to the F-actin ring and required in its formation. They are
thought to be involved in a late step of the F-actin ring assembly. An
IQGAP-like protein has also been considered to be involved in a late step of
the F-actin ring assembly (Chang et al.,
1996
; Eng et al.,
1998
).
At the onset of cytokinesis, Spg1, a small GTP-binding protein, regulates
initiation of F-actin ring contraction or septum formation, or both, through
controlling localization and function of downstream effectors, Cdc7 and Sid2
protein kinases (Schmidt et al.,
1997; Sohrmann et al.,
1998
; Sparks et al.,
1999
). It has been reported that the septum formation never occurs
in mutants of these genes, although the F-actin ring is formed during mitosis
(Fankhauser and Simanis, 1994
;
Schmidt et al., 1997
;
Balasubramanian et al.,
1998
).
We are interested in the molecular process of the early step of the F-actin ring formation. From the above results, two distinct events seem to occur in the early step of the F-actin ring formation: one is accumulation of Mid1 followed by the accumulation of F-actin as the medial broad ring, and then the ring narrows. The other is positioning of the Cdc12 as a spot in the medial cortex. The spot then forms the ring by extending a strand. However, the relationship between these events remains unclear. Furthermore, little is known about the process of structural rearrangement of F-actin during the F-actin ring formation including how actin filaments are arranged in the `broad F-actin ring'. Thus, we investigated the process of the F-actin ring formation by optical sectioning and three-dimensional (3D) microscopy in the wild-type S. pombe cells and some cytokinesis mutant cells. Our observations suggest that the F-actin ring formation is initiated by the accumulation of the F-actin cables and the formation of an aster-like structure composed of F-actin cables at the cell equator during prophase.
![]() |
Materials and Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Fluorescence microscopy
Immunostaining was performed as previously described
(Arai et al., 1998). Mitotic
stages were defined by localization of spindle pole bodies (SPBs) according to
Hagan (Hagan, 1998
).
Representative images are shown in the following figures. The SPBs were
labeled with rabbit anti-Sad1 antibody (a gift from Iain Hagan) and then with
rhodamine-conjugated anti-rabbit IgG antibody (Cappel, Durham, NC). For
F-actin and nuclear staining, Bodipy
(4,4-difluoro-4-bora-3a,4a-diaza-s-indacene)-phallacidin (Molecular Probes
Inc., Eugene, OR) and 4',6-diamidino phenylindole (DAPI) were used,
respectively. For usual observations, the specimens were examined under a
Zeiss Axioscope fluorescence microscope using a Plan Apochromat 63x lens
and then photographed on Kodak T-Max films of ASA 400
(Fig. 6a-h). To obtain
deconvoluted optical sections and 3D reconstructed images, we used a Delta
Vision system (Applied Precision Inc., Issaquah, WA) with an Olympus IX70
fluorescence microscope equipped with a UPlanApo 100x lens according to
the manufacturer's protocol (Figs
1,2,3,4,5,6,7,8,9).
Original fluorescence micrographs of serial optical sections were taken every
0.2 µm in the direction of Z-axis and digitized. The original images were
deconvoluted, and then reconstructed to obtain 3D images of the cells. For the
3D reconstruction, we examined two calculation modes, `Max Intensity' and
`Additive'. In the former mode, major fluorescences were emphasized but minor
ones were removed. The minor fluorescences were retained, however, in the
latter mode. Because the Additive mode was suitable for visualization of
F-actin structures, especially continuous F-actin cables, we used this mode to
show whole F-actin cables in 3D images in this paper. To visualize F-actin
structures more in detail, we showed deconvoluted serial sections.
|
|
|
|
|
|
|
|
|
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Accumulation of F-actin cables at the medial region in wild-type
cells during early mitosis
An interphase cell that passed through `new end take off'
(Marks and Hyams, 1985) showed
a single spot of SPB (Fig. 1).
In this stage, several F-actin cables ran in the longitudinal direction of the
cell from one end of the cell to the other, and several short F-actin cables
were also observed. F-actin patches were concentrated in both growing ends and
at least one end of each F-actin cable was linked to an F-actin patch.
In the cells showing two SPB spots in one optical section
(Fig. 2), a few major F-actin
cables were seen to adjoin the SPBs at the mid region of the cells and branch
in this region. The stage of these cells was considered to be pre-prophase to
early prophase, for the following reasons. First, the length of these cells
was in the range of 13-14 µm, which was the size of mitotic cells we used.
Second, two adjacently positioned SPB spots were recognized in these cells in
a 0.2 µm section. Third, maturation of duplicated SPBs, which are not
separated yet, occurs in S. pombe cells during this stage, and the
size of one matured SPB is about 0.2 µm
(Ding et al., 1997). Although
most of the F-actin patches were localized near the ends, a considerable
number were seen in the cytoplasm away from the ends. Some of them were
attached to the F-actin cables.
In a metaphase cell (Fig. 3), the density of the F-actin cables increased at the medial region of the cell. Most of these F-actin cables seemed to emanate radially from a focus forming an aster-like structure, whereas there were some cables that were not involved in this structure. A single F-actin cable extends from the aster and encircles the cell at the equator. We call this F-actin cable a `leading F-actin cable'. In Fig. 3A, this cable once branched into two cables and then they merged again. Two leading cables extending from the aster and moving in two opposite directions were occasionally seen (Fig. 3B). At the reverse side of the medial cortex, a clear longitudinal cable was often observed (Fig. 3). Serial sectioning showed that the accumulation of the F-actin cables and the formation of the aster-like structure occur near the cell surface but not in the inner cytoplasm.
Association of F-actin cables with the F-actin ring
In a cell at anaphase A (Fig.
4), a thicker F-actin ring was observed. A fairly distorted
portion is seen at one side of the F-actin ring in contrast to the portion at
the other side of the ring (Fig.
4d-f). This distorted portion may have been the aster-like
structure at the previous stage. Heavy accumulation of F-actin cables were
seen at the medial region, and they were linked to the F-actin ring. In a cell
in anaphase B (Fig. 5a), a
sharp and `straight' F-actin ring was seen, to which some F-actin cables were
always linked. In a late anaphase cell
(Fig. 5b) in which SPBs were
completely separated towards both ends, an F-actin ring accompanied by fewer
F-actin cables was seen.
The F-actin patches showed relatively random distribution during prophase to mid anaphase, being sparse at the mid region as compared with interphase. However, the F-actin patches were scarcely seen, especially at the medial region of the cell during late anaphase.
As the cells initiated cytokinesis (Fig. 5c), the F-actin ring started to contract, and the F-actin cables became clearly visible again extending from the division site. The F-actin patches also reappeared at the medial region of the cells (Fig. 5c). In either a late cytokinesis cell (Fig. 5d) or separating daughter cells (Fig. 5e), many F-actin patches emerged around the newly formed septa, and the F-actin cables elongated from the septa. Serial sections showed that the F-actin cables were linked to either the shrinking ring or the F-actin patches at the medial region (data not shown).
Broad F-actin ring in nda3 conditional mutant
The broad F-actin ring similar to the one seen in the wild-type cells
during prophase (Arai et al.,
1998) has also been observed in the nda3 cold-sensitive
mutant at a restrictive temperature (Chang
et al., 1996
). This mutant is thought to be arrested at prophase,
and duplicated SPBs cannot separate from each other on the nucleus
(Hiraoka et al., 1984
;
Kanbe et al., 1990
). At 2-4
minutes after release from the arrested condition, the SPBs initiate
separation on the nucleus, and then chromosome segregation (
10 minutes
after the release) and cytokinesis (
14 minutes after the release) proceed
in order (Hiraoka et al.,
1984
; Kanbe et al.,
1990
). However, it has not yet been examined how this broad
F-actin ring is related to that seen in the wild-type cells and reorganization
of the actin cytoskeleton after the release.
We observed localization of F-actin in both the arrested nda3 mutant cells and the released cells using Bodipy-phallacidin staining. In the arrested cells showing condensed chromosomes, a broad F-actin ring and some elongated cables were seen as observed by conventional fluorescence microscopy (Fig. 6a,b). As analyzed by the optical sectioning and 3D reconstitution microscopy, an F-actin ring accompanied by several F-actin cables was visualized in the arrested cells (Fig. 6i). This appearance is very similar to that of early anaphase cell. These rings seemed to be loosely packed as compared to those seen in released cells (see below).
The F-actin rings were narrowed at 4 minutes after shift to permissive
temperature (Fig. 6c,d). As
analyzed by the 3D reconstruction microscopy
(Fig. 6j), packed F-actin rings
were clearly visible. These rings did not seem to be accompanied by the
F-actin cables. F-actin patches appeared all over the cell cortex. After the
completion of the chromosome segregation
(Fig. 6e-h), the F-actin ring
started to contract at 10 minutes after the release.
Actin cytoskeleton in several temperature-sensitive cytokinesis
mutants
Because we considered that cytokinesis mutants might have defects in
organization of the F-actin cables, we examined actin cytoskeleton in some
temperature-sensitive mutant cells. We examined the second mitosis after
temperature shift to observe the exact phenotype of the mutant cells. Detailed
mitotic stages were determined both by the extent of condensation of
chromosomes and the position of the SPBs on the nuclei. During anaphase, two
sister nuclei move towards both ends of the cell. However, they are pulled
back towards the cell center after anaphase, and a septum is formed between
the closely located nuclei during cytokinesis in the wild-type cell
(Hagan, 1998). The SPBs always
localize on the moving side of the nucleus. In most of cytokinesis mutant
cells examined in this study, the sister nuclei separated after the first
mitosis at a restrictive temperature were located close to each other near the
center of the elongated cells. They stayed in these positions without a septum
during the next interphase to metaphase. As a result, two spindles tended to
elongate parallel to each other during the following anaphase in the second
mitotic cycle.
F-actin structures in cdc12 and cdc15 mutant cells were analyzed by the optical sectioning microscopy. During interphase, F-actin cables seemed to be normally arranged in both the cdc12 and the cdc15 mutant cells (data not shown). In a cdc12-112 mutant cell at metaphase (Fig. 7A), two aster-like structures were seen in the medial cell cortex. However, no leading F-actin cable was recognized. During anaphase (Fig. 7B), the aster-like structures were still recognizable and F-actin cables were accumulated at the medial region. However, again, no leading F-actin cable was seen, in contrast to anaphase wild-type cells in which the aster-like structure could not clearly be recognized; instead, a distorted (Fig. 4) or a packed (Fig. 5a) F-actin ring was observed. After anaphase (Fig. 7C), some F-actin cables were longitudinally elongated like interphase wild-type cells, and the accumulation of the F-actin cables at the medial region was not seen any longer.
In a cdc15-140 mutant cell at metaphase (Fig. 8A), two aster-like structures, each possessing a leading F-actin cable, were seen. During anaphase (Fig. 8B), two distorted F-actin rings, which seemed to be partially connected with each other, were formed. After anaphase (Fig. 8C), several longitudinal F-actin cables were clearly seen. These cables were elongated from a point at the medial cell cortex. A remnant of an F-actin ring, which did not seem to have shrunk after anaphase, was also seen at the cell equator, and it was linked to the medial point from which the F-actin cables were elongated. In the first mitotic cycle after shift to the restrictive temperature, only one aster-like structure was observed both in the cdc12 and cdc15 mutant cells (data not shown). Therefore, it is confirmed that one nucleus formed one aster-like structure, and that the two aster-like structures seen during the second mitosis were not fragments derived from one single aster-like structure through its degradation.
We further analyzed F-actin structures in spg1, cdc7 and sid2 mutant cells during and after the F-actin ring formation. These mutant cells showed similar phenotypes. During metaphase, we saw two aster-like structures near the nuclei. A leading cable seemed to extend from each of the aster-like structures (data not shown). Then, a single F-actin ring was formed in about half of the cells at late anaphase (Fig. 9a,b), whereas double rings were also observed in the remainder (Fig. 9c,d). We could never find cells that had no F-actin ring in this stage. Most of the double rings seemed to be connected to each other at one point. Moreover, one of the double rings was a complete ring, whereas the other one was incomplete (Fig. 9d). In the first division cycle after shift to the restrictive temperature, only one single F-actin ring was observed in these mutant cells during anaphase (data not shown). After anaphase, 24 out of 34 spg1 mutant cells, 22 out of 31 cdc7 mutant cells and 6 out of 32 sid2 mutant cells still possessed the F-actin ring which did not seem to have contracted (Fig. 9g,h, right cell) whereas the rest of the cells did not (Fig. 9e,f). As the SPBs were pulled back further towards the cell center, the persisted F-actin ring disappeared (Fig. 9g,h, left cell). Being distinct from the cdc15 cells at the same stage, the longitudinal F-actin cables were not seen in these mutant cells. Curiously, F-actin patches reappeared at the medial region (Fig. 9f,h), although septum formation was never observed in these cells.
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Protein constituents other than actin in these structures are reported to
be different. Tropomyosin is present in both the F-actin cables and the
F-actin ring, but is absent in the majority of the F-actin patches
(Arai et al., 1998). By
contrast, Arp3 is present in the F-actin patches but is absent from both the
F-actin cable and the F-actin ring
(McCollum et al., 1996
;
Arai et al., 1998
). Type II
myosin light chains (Cdc4 and Rlc1)
(McCollum et al., 1995
;
Le Goff et al., 2000
;
Naqvi et al., 2000
), type II
myosin heavy chains (Kitayama et al.,
1997
; May et al.,
1997
; Motegi et al.,
2000
) and an IQGAP-like protein Rng2
(Eng et al., 1998
) are present
in the F-actin ring but not in the F-actin patches and cables.
-actinin
(Ain1) (Wu et al., 2001
) and
fimbrin (Fim1) (Wu et al.,
2001
; Nakano et al.,
2001
) are present in the F-actin ring and in the patches but not
in the cables. These specific components may characterize and define
organization and functions of the three F-actin cytoskeletons in S.
pombe.
Formation of aster-like structure of F-actin cables
Major F-actin cables were branched near the SPBs at early prophase, and
then an aster-like structure composed of the F-actin cables was seen in the
middle region during metaphase. The branching of the F-actin cables seemed to
be a first step in the formation of the aster-like structure. Because these
events occur at a position very close to the SPBs, which are not yet separated
from each other, it is tempting to speculate that the nonseparated SPBs
control the formation of the aster-like structure during early prophase. The
formation of this structure is a novel finding in S. pombe during
early mitosis. It has also been reported that radial F-actin arrays, which are
similar to the aster-like structure described here, are observed at a future
site of new hypha formation and at a future elongation tip of each spore in
the oomycete Saprolegnia ferax
(Bachewich and Heath, 1998).
The relationship between this structure and the SPBs/nucleus, however, has not
been investigated.
Accumulation of F-actin cables at the medial region of the cell
during metaphase
A broad and faint F-actin ring has been observed in early mitotic wild-type
cells using conventional fluorescence microscopy
(Chang et al., 1996;
Arai et al., 1998
;
Bähler et
al., 1998
). In this study, the density of the F-actin cables
increased in this region in metaphase wild-type cells. Thus, it is reasonable
to consider that the broad F-actin ring visualized by the conventional
microscopy actually represents the accumulated F-actin cables. Where do these
F-actin cables come from? The first possibility is that the F-actin cables are
originated from the center of the aster-like structure. However, our
observation suggests that not all the cables emanated from the aster-like
structure. A second possibility is that actin polymerization and subsequent
bundling might have occurred in the broad medial region. However, we could not
detect newly polymerized actin filaments, not yet bundled, in the metaphase
wild-type, as analyzed by optical sectioning and 3D reconstruction microscopy.
Thus, this theory is weakened, although the polymerized actin filaments could
be removed from the image as background by the deconvolution process and only
the strongly fluorescent F-actin cables could have remained. The third
possibility is that the F-actin cables move from both sides of the cell to the
medial region. This is probable because the density of the F-actin cables move
from the ends to the medial region as mitosis progresses. It is necessary to
live-record the movement of the cables to prove this hypothesis and to know
how the cables move in the cell by using expression of GFP-actin fusion
protein, for example.
Process of F-actin ring formation
A schematic model for the F-actin ring formation is presented in
Fig. 10, which is based on the
present observations. In this model, it is supposed that a positional
signal(s) (see below) is generated from the SPBs or nucleus, or both, and
reaches the cortex of the medial region of the cell at pre-prophase. The
signal(s) induces the formation of the aster-like structure during early
prophase. The accumulation of the F-actin cables is initiated during
prophase.
|
During the formation of the aster-like structure, a leading F-actin cable seems to extend from this structure, and encircle the cell at the equator to form the primary F-actin ring during metaphase. F-actin cables accumulated in the medial region seem to be connected to the leading cable during early anaphase and then all the cable structures are packed to form the complete F-actin ring during late anaphase.
It has been shown that the type II myosin heavy chain Myo2 accumulates
around the mid region independently of its head domain
(Naqvi et al., 1999) and
F-actin (Motegi et al., 2000
)
during early mitosis. Myo2 forms spot structures that do not colocalize with
the aster-like structure. Subsequently, the Myo2 spots change into a
spot-fiber network, and then the network is packed to form the ring along with
the packing of the F-actin cables to form the F-actin ring. Colocalization of
Myo2 with the F-actin cable is seen during this stage
(Motegi et al., 2000
), and
this process may require ATPase activity of Myo2
(Naqvi et al., 1999
) and the
presence of F-actin (Motegi et al.,
2000
).
Conversely, F-actin cables can accumulate in the mid region of the cell in
the cdc4 mutant at the restrictive temperature
(Motegi et al., 2000),
although both Cdc4 (McCollum et al.,
1995
) and myosin II heavy chains (Myo2 and Myp2/Myo3)
(Motegi et al., 1997
;
Motegi et al., 2000
) are
required for the formation of the F-actin ring. These observations suggest
that the changes in F-actin organization at the mid region of the cell, which
include the accumulation of F-actin cables and formation of the aster-like
structure, and changes in myosin II spots at the mid region involve processes
that are independent of each other. The next step to complete the F-actin ring
formation may require the interaction of these structures.
Positional signals and the aster-like structure
Recently, it has been proposed that the positional signal(s) for the
F-actin ring formation is generated from the nucleus in the middle of the cell
(Chang and Nurse, 1996) and
that transduction of such signal(s) may be mediated by SPBs during prophase
(Bähler et
al., 1998
) in S. pombe. In fact, it has been reported
that Mid1, which has been considered to position the site of the F-actin ring
formation (Chang et al., 1996
;
Sohrmann et al., 1996
),
localizes on the nuclear membrane during interphase and relocalizes to the
broad medial region of the cells during prophase
(Sohrmann et al., 1996
;
Bähler et
al., 1998
). For this relocalization, function of Plo1 is required,
and Plo1 localizes to the SPBs during prophase to anaphase
(Bähler et
al., 1998
). Therefore, these factors are probably involved in the
signaling pathway for the F-actin ring formation. However, it has not been
clarified how the signal(s) is transferred from the nucleus or the SPBs, or
both, to the cortex of the cell. Although the SPBs are attached on the nuclear
membrane during interphase, they are buried in the nuclear membrane during
mitosis in S. pombe: a part of the nuclear membrane just beneath the
SPBs is collapsed at pre-prophase and then the SPBs are settled in the nuclear
membrane (Ding et al., 1997
).
The partial collapse of the nuclear membrane at pre-prophase may be a trigger
to generate signals for initiation of the F-actin ring formation. The unknown
signal(s) from the SPBs may mark a spot on the medial cortex at pre-prophase
or early prophase, and the formation of the aster-like structure may be
initiated from this spot. The accumulated F-actin cables that are not involved
in the aster-like structure may be regulated by other signals that localize to
the broad medial region during prophase to anaphase, such as Mid1.
Possible role of Cdc12 in the ring formation
It has been reported that overexpressed Cdc12-GFP forms a spot and this
spot moves in the cell during interphase, probably along microtubular tracks
and F-actin tracks. The spot arrives at the medial region of the cell during
prophase and a ring of Cdc12-GFP is formed from it by the spot extending a
Cdc12-GFP strand along the cell equator during prophase to metaphase
(Chang et al., 1997;
Chang, 1999
). It has also been
reported that a domain of Cdc12 possesses an ability to interact with
components of SPBs (Petersen et al.,
1998
). It is possible that the Cdc12-GFP spot mediates the
positional signal(s) from the SPBs to the medial cortex, or that the Cdc12-GFP
spot recognizes the signal(s) that had been transferred on the medial cortex,
and becomes the center of the aster-like structure. By contrast, this
structure was observed in cdc12 mutant cells during prophase to
metaphase in this study, suggesting that Cdc12 may not be necessary in the
formation of the aster. However, it could be that the mutated Cdc12 still
possesses function to form the aster-like structure. However, no leading
F-actin cable was observed in the cdc12 mutant cells during mitosis,
although the accumulation of the F-actin cables around the mid region
occurred. This suggests that Cdc12 is required to form the leading F-actin
cable and thereby to form the primary F-actin ring. The formation of the
strand that encircles the cell equator from the Cdc12-GFP spot seems to be
similar to the leading cable formation from the aster-like structure. Thus,
the leading F-actin cable may be formed by being influenced by the Cdc12
strand. It has been reported that Cdc12 interacts with Cdc3 profilin
(Chang et al., 1997
), and
profilin is concentrated around the mid region during mitosis
(Balasubramanian et al., 1994
).
Actin polymerization may be stimulated by Cdc12 via profilin in the formation
of both the aster and the leading F-actin cable. It will be necessary in the
future to show actual elongation of the leading F-actin cable from the
aster-like structure and its relationship to the elongation of the Cdc12
strand in living cells by means of expression of GFP-fusion proteins. In
addition, an activity of the accumulation of the F-actin cables in the mid
region is retained during prophase to anaphase, and this accumulation is not
very marked after anaphase in the cdc12 mutant cells. This transition
of the F-actin cable arrangement does not seem to require the Cdc12
function.
Rng2, an IQGAP-like protein, has been considered to play an important role
in the signaling of the F-actin ring formation, as it localizes to the SPBs
during interphase, and relocalizes to the medial ring during mitosis
(Eng et al., 1998).
rng2 null cells form an F-actin spot containing Cdc3 and calmodulin
at the mid region, although they cannot form the F-actin ring. This spot
formation is thought to be an intermediate step in the pathway of the F-actin
ring formation (Eng et al.,
1998
). Moreover, rng2 mutation shows synthetic lethality
with cdc12. Therefore, it would be important to investigate how Cdc12
and Rng2 are involved in the formation of the aster-like structure, and
whether this structure and the F-actin spot are related.
Arrangement of F-actin cables in cdc15 mutant
In cdc15-140 mutant cells, the aster-like structure, the leading
cable and the distorted F-actin ring were almost normally formed in order
during mitosis, although cytokinesis never occurred subsequently. It has also
been reported that the F-actin ring is formed in both cdc15-140 and
cdc15-A5 mutant cells during mitosis, although both recruitment of
the F-actin patches to the medial region and septation after the F-actin ring
formation do not occur (Balasubramanian et
al., 1998). On the contrary, it has been reported that the F-actin
ring is formed only in a small population of mitotic cells of
cdc15-140 and cdc15-287
(Fankhauser et al., 1995
;
Chang et al., 1997
). Three
different cdc15 mutant strains were used in these reports, including
the present study. It may be that both the different mutations and minute
differences in experimental conditions have resulted in the appearance of
different phenotypes. In addition, the distorted F-actin ring may not have
been regarded as the F-actin ring by some microscopic systems used for the
above observation. From our detailed observation, the cdc15-140
mutant could form the distorted F-actin ring during anaphase, but the
subsequent packing of the ring and its contraction did not occur. It is
suggested that Cdc15 is required for packing of the accumulated F-actin cables
to form the complete F-actin ring, which is able to contract.
In the cdc15-140 cells, a part of the F-actin ring still remained
after anaphase, suggesting that these cells also have defects in the
disassembly of the F-actin ring after anaphase. It has been reported that
Cdc15 has a structural similarity to Imp2, which is required for F-actin ring
disassembly and cell separation, and that a double mutant of
cdc15-140 and imp2 shows synthetic lethality
(Demeter and Sazer, 1998).
These data suggest that Cdc15 is required for the rearrangements of actin
cytoskeleton in the process of cytokinesis, including the packing of the
F-actin cables during the formation of the F-actin ring and disassembly of the
F-actin ring after the contraction.
Arrangement and function of F-actin ring in nda3 mutant
In the nda3 mutant, the formation of the F-actin ring and the
accumulation of fine F-actin cables associated with the ring were observed at
the restrictive temperature. The nda3 mutant cells are thought to be
arrested at prophase, as duplicated SPBs cannot initiate separation on the
nucleus because microtubules cannot elongate between these SPBs in this mutant
(Hiraoka et al., 1984;
Kanbe et al., 1990
). However,
in the arrested nda3 mutant cells, we could not detect the aster-like
structure, which is normally formed during prophase in the wild-type cell, and
the F-actin ring was similar to that of wild-type cells in early anaphase.
Therefore, it could be that the mechanism by which the F-actin ring formation
is controlled proceeded beyond prophase to early anaphase; the activity to
accumulate the F-actin cables is probably stimulated and retained once cells
enter prophase. As soon as the arrested cells were shifted to the permissive
temperature, the tightly packed F-actin ring was formed, which was similar to
the complete F-actin ring observed in the wild-type cells at late anaphase,
which then contracted and cytokinesis progressed. Interestingly, the F-actin
ring in the arrested nda3 mutant cells never contracted unless
temperature was increased. These results may suggest that the completion of
the F-actin ring formation is an essential step for the cell to enter
cytokinesis, and that function of cytoplasmic microtubules is required for
this process. However, nda3 mutant cells with mad2 defect
can undergo septation (He et al.,
1997
). Because Mad2 has been known as a component of spindle check
point (Li and Murray, 1991
;
He et al., 1997
), the
completion of the F-actin ring formation and the initiation of the ring
contraction may be regulated by such a mitotic check point.
F-actin ring formation during cytokinesis in septum initiation
mutants
At late anaphase, the single or double F-actin rings were formed in the
spg1, cdc7 or sid2 mutant cells. In the latter case, one
complete ring and one incomplete ring were connected to each other at one
point. It is possible that the single ring was formed as a result of fusion of
the double rings. The leading F-actin cable, the aster-like structure and the
F-actin ring are formed in these mutant cells, indicating that the components
of the septum initiation network (McCollum
and Gould, 2001) are not involved in the formation of the F-actin
ring.
Spg1 localizes only to the SPB (s) at all stages of the cell cycle and
recruits Cdc7 to the SPB(s) during mitosis
(Schmidt et al., 1997;
Sohrmann et al., 1998
).
However, Sid2 localizes not only to the SPBs but also to the F-actin ring and
both sides of the forming septum during late anaphase to cytokinesis
(Sparks et al., 1999
). It has
also been reported that Sid2 is required for initiation of the F-actin ring
contraction or septum formation, or both, as a downstream effector of the
Spg1-Cdc7 pathway (Balasubramanian et al.,
1998
; Sparks et al.,
1999
). In fact, septum formation never occurred in these septum
initiation mutant cells. We observed that the majority of the spg1 or
cdc7 mutant cells possessed the F-actin ring after anaphase, whereas
the ring disappeared earlier in most of the sid2 mutant cells. Thus,
Sid2 may contribute to retaining the F-actin ring structure during contraction
through localizing to this structure.
![]() |
Acknowledgments |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Alfa, C. and Hyams, J. (1990). Distribution of tubulin and actin through the cell division cycle of the fission yeast Schizosaccharomyces japonicus var. versatilis: a comparison with Schizosaccharomyces pombe. J. Cell Sci. 96, 71-77.[Abstract]
Arai, R., Nakano, K. and Mabuchi, I. (1998). Subcellular localization and possible function of actin, tropomyosin and actin-related protein 3 (Arp3) in the fission yeast Schizosaccharomyces pombe. Eur. J. Cell Biol. 76,288 -295.[Medline]
Bachewich, C. and Heath, I. B. (1998). Radial
F-actin arrays precede new hypha formation in Saprolegnia:
implications for establishing polar growth and regulating tip morphogenesis.
J. Cell Sci. 111,2005
-2016.
Bähler, J., Steever, A. B.,
Wheatley, S., Wang, Y. L., Pringle, J. R., Gould, K. L. and McCollum, D.
(1998). Role of polo kinase and Mid1p in determining the site of
cell division in fission yeast. J. Cell Biol.
143,1603
-1616.
Balasubramanian, M. K., Helfman, D. M. and Hemmingsen, S. M. (1992). A new tropomyosin essential for cytokinesis in the fission yeast S. pombe. Nature 360, 84-87.[Medline]
Balasubramanian, M. K., Hirani, B. R., Burke, J. D. and Gould, K. L. (1994). The Schizosaccharomyces pombe cdc3+ gene encodes a profilin essential for cytokinesis. J. Cell Biol. 125,1289 -1301.[Abstract]
Balasubramanian, M. K., McCollum, D., Chang, L., Wong, K. C. Y.,
Naqvi, N. I., He, X., Sazer, S. and Gould, K. L. (1998).
Isolation and characterization of new fission yeast cytokinesis mutants.
Genetics 149,1265
-1275.
Bezanilla, M., Forsburg, S. L. and Pollard, T. D.
(1997). Identification of a second myosin-II in
Schizosaccharomyces pombe: Myp2p is conditionally required for
cytokinesis. Mol. Biol. Cell
8,2693
-2705.
Chang, F. (1999). Movement of a cytokinesis factor cdc12p to the site of cell division. Curr. Biol. 9,849 -852.[Medline]
Chang, F. and Nurse, P. (1996). How fission yeast fission in the middle. Cell 84,191 -194.[Medline]
Chang, F., Woollard, A. and Nurse, P. (1996).
Isolation and characterization of fission yeast mutants defective in the
assembly and placement of the contractile actin ring. J. Cell
Sci. 109,131
-142.
Chang, F., Drubin, D. and Nurse, P. (1997).
cdc12p, a protein for cytokinesis in fission yeast, is a component of the cell
division ring and interacts with profilin. J. Cell
Biol. 137,169
-182.
Demeter, J. and Sazer, S. (1998). imp2, a new
component of the actin ring in the fission yeast Schizosaccharomyces
pombe. J. Cell Biol. 143,415
-427.
Ding, R., West, R. R., Morphew, M., Oakley, B. R. and McIntosh, J. R. (1997). The spindle pole body of Schizosaccharomyces pombe enters and leaves the nuclear envelope as the cell cycle proceeds. Mol. Biol. Cell 8,1461 -1479.[Abstract]
Edamatsu, M. and Toyoshima, Y. Y. (1996). Isolation and characterization of pos mutants defective in correct positioning of septum in Schizosaccharomyces pombe. Zool. Sci. 13,235 -239.[Medline]
Eng, K., Naqvi, N. I., Wong, K. C. Y. and Balasubramanian, M. K. (1998). Rng2, a protein required for cytokinesis in fission yeast, is a component of the actomyosin ring and the spindle pole body. Curr. Biol. 8,611 -621.[Medline]
Fankhauser, C. and Simanis, V. (1994). The cdc7 protein kinase is a dosage dependent regulator of septum formation in fission yeast. EMBO J. 13,3011 -3019.[Abstract]
Fankhauser, C., Reymond, A., Cerutti, L., Utzig, S., Hofmann, K. and Simanis, V. (1995). The S. pombe cdc15 gene is a key element in the reorganization of F-actin at mitosis. Cell 82,435 -444.[Medline]
Hagan, I. (1998). The fission yeast microtubule
cytoskeleton. J. Cell Sci.
111,1603
-1612.
He, X., Patterson, T. E. and Sazer, S. (1997).
The Schizosaccharomyces pombe spindle checkpoint protein mad2p blocks
anaphase and genetically interacts with the anaphase-promoting complex.
Proc. Natl. Acad. Sci. USA
94,7965
-7970.
Hiraoka, Y., Toda, T. and Yanagida, M. (1984). The NDA3 gene of fission yeast encodes beta-tubulin: a cold-sensitive nda3 mutation reversibly blocks spindle formation and chromosome movement in mitosis. Cell 39,349 -358.[Medline]
Ishiguro, J. (1998). Genetic control of fission yeast cell wall synthesis: the genes involved in wall biogenesis and their interactions in Schizosaccharomyces pombe. Genes Genet. Syst. 73,181 -191.[Medline]
Ishiguro, J. and Kobayashi, W. (1996). An actin point-mutation neighboring the `hydrophobic plug' causes defects in the maintenance of cell polarity and septum organization in the fission yeast Schizosaccharomyces pombe. FEBS Lett. 392,237 -241.[Medline]
Kanbe, T., Hiraoka, Y., Tanaka, K. and Yanagida, M. (1990). The transition of cells of the fission yeast ß-tubulin mutant nda3-311 as seen by freeze-substitution electron microscopy. J. Cell Sci. 96,275 -282.[Abstract]
Karpova, T., McNally, J., Moltz, S. and Cooper, J.
(1998). Assembly and function of the actin cytoskeleton of yeast:
relationships between cables and patches. J. Cell
Biol. 142,1501
-1517.
Kitayama, C., Sugimoto, A. and Yamamoto, M.
(1997). Type-II myosin heavy chain encoded by the myo2
gene composes the contractile ring during cytokinesis in
Schizosaccharomyces pombe. J. Cell Biol.
137,1309
-1319.
Kobori, H., Yamada, N., Taki, A. and Osumi, M. (1989). Actin is associated with the formation of the cell wall in reverting protoplasts of the fission yeast Schizosaccharomyces pombe.J. Cell Sci. 94,635 -646.[Abstract]
Le Goff, X., Utzig, S. and Simanis, V. (1999). Controlling septation in fission yeast: finding the middle, and timing it right. Curr. Genet. 35,571 -584.[Medline]
Le Goff, X., Motegi, F., Salimova E., Mabuchi, I. and Simanis,
V. (2000). The S. pombe rlc1 gene encodes a putative
myosin regulatory light chain that binds the type II myosins myo3p and myo2p.
J. Cell Sci. 113,4157
-4163.
Li, R. and Murray, A. W. (1991). Feedback control of mitosis in budding yeast. Cell 66,519 -531.[Medline]
Mabuchi, I. (1986). Biochemical aspects of cytokinesis. Int. Rev. Cytol. 101,175 -213.[Medline]
Mabuchi, I. (1994). Cleavage furrow: timing of
emergence of contractile ring actin filaments and establishment of the
contractile ring by filament bundling in sea urchin eggs. J. Cell
Sci. 107,1853
-1862.
Marks, J. and Hyams, J. S. (1985). Localization of F-actin through the cell division cycle of Schizosaccharomyces pombe.Eur. J. Cell Biol. 39,27 -32.
May, K. M., Watts, F. Z., Jones, N. and Hyams, J. S. (1997). Type II myosin involved in cytokinesis in the fission yeast, Schizosaccharomyces pombe. Cell Motil. Cytoskeleton 38,385 -396.[Medline]
McCollum, D. and Gould, K. L. (2001). Timing is everything: regulation of mitotic exit and cytokinesis by the MEN and SIN. Trends Cell Biol. 11,89 -95.[Medline]
McCollum, D., Balasubramanian, M. K., Pelcher, L. E. and Hemmingsen, S. M. (1995). Schizosaccharomyces pombe cdc4+ gene encodes a novel EF-hand protein essential for cytokinesis. J. Cell Biol. 130,651 -660.[Abstract]
McCollum, D., Feoktistova, A., Morphew, M., Balasubramanian, M. K. and Gould, K. L. (1996). The Schizosaccharomyces pombe actin-related protein, Arp3, is a component of the cortical actin cytoskeleton and interacts with profilin. EMBO J. 15,6438 -6446.[Abstract]
Motegi, F., Nakano, K., Kitayama, C., Yamamoto, M. and Mabuchi, I. (1997). Identification of Myo3, a second type-II myosin heavy chain in the fission yeast Schizosaccharomyces pombe. FEBS Lett. 420,161 -166.[Medline]
Motegi, F., Nakano, K. and Mabuchi, I. (2000).
Molecular mechanism of myosin-II assembly at the division site in
Schizosaccharomyces pombe. J. Cell Sci.
113,1813
-1825.
Nakano, K., Satoh, K., Morimatsu, A., Ohnuma, M. and Mabuchi,
I. (2001). Interactions among a fimbrin, a capping protein,
and an actin-depolymerizing factor in organization of the fission yeast actin
cytoskeleton. Mol. Biol. Cell
12,3515
-3526.
Naqvi, N. I., Eng, K., Gould, K. L. and Balasubramanian, M.
K. (1999). Evidence for F-actin-dependent and independent
mechanisms involved in assembly and stability of the medial actomyosin ring in
fission yeast. EMBO J.
18,854
-862.
Naqvi, N. I., Wong, K. C. Y., Tang, X. and Balasubramanian M. K. (2000). Type II myosin regulatory light chain relieves auto-inhibition of myosin-heavy-chain function. Nat. Cell Biol. 2,855 -858.[Medline]
Ohkura, H., Hagan, I. M. and Glover, D. M. (1995). The conserved Schizosaccharomyces pombe plo1, required to form a bipolar spindle, the actin ring, and septum, can drive septum formation in G1 and G2 cells. Genes Dev. 9,1059 -1073.[Abstract]
Pelham, R. J., Jr and Chang, F. (2001). Role of actin polymerization and actin cables in actin-patch movement in Schizosaccharomyces pombe. Nat. Cell Biol. 3, 235-244.[Medline]
Petersen, J., Nielsen, O., Egel, R. and Hagan, I. M.
(1998). FH3, a domain found in formins, targets the fission yeast
formin Fus1 to the projection tip during conjugation. J. Cell
Biol. 141,1217
-1228.
Rappaport, R. (1986). Establishment of the mechanism of cytokinesis in animal cells. Int. Rev. Cytol. 101,245 -281.
Schmidt, S., Sohrmann, M., Hofmann, K., Woollard, A. and Simanis, V. (1997). The Spg1p GTPase is an essential dosage-dependent inducer of septum formation in Schizosaccharomyces pombe.Genes Dev. 11,1519 -1534.[Abstract]
Sohrmann, M., Fankhauser, C., Brodbeck, C. and Simanis, V. (1996). The dmf1/mid1 gene is essential for correct positioning of the division septum in fission yeast. Genes Dev. 10,2707 -2719.[Abstract]
Sohrmann, M., Schmidt, S., Hagan, I. and Simanis, V.
(1998). Asymmetric segregation on spindle poles of the
Schizosaccharomyces pombe septum-inducing protein kinase Cdc7p.
Genes Dev. 12,84
-94.
Sparks, C. A., Morphew, M. and McCollum, D.
(1999). Sid2p, a spindle pole body kinase that regulates the
onset of cytokinesis. J. Cell Biol.
146,777
-790.
Wu, J. Q., Bähler, J. and Pringle,
J. R. (2001). Roles of a fimbrin and an alpha-actinin-like
protein in fission yeast cell polarization and cytokinesis. Mol.
Biol. Cell 12,1061
-1077.