Download Decision of Spindle Poles and Division Plane by Double

Plant Cell Physiol. 46(3): 531–538 (2005)
doi:10.1093/pcp/pci055, available online at www.pcp.oupjournals.org
JSPP © 2005
Decision of Spindle Poles and Division Plane by Double Preprophase Bands in
a BY-2 Cell Line Expressing GFP–Tubulin
Arata Yoneda 1, Minori Akatsuka 1, Hidemasa Hoshino, Fumi Kumagai and Seiichiro Hasezawa 2
Department of Integrated Biosciences, Graduate School of Frontier Sciences, The University of Tokyo, Kashiwanoha 5-1-5, Kashiwa, Chiba
Prefecture, 277-8562 Japan
;
ever, as the PPB disappears well before cytokinesis, it has been
suggested that some form of ‘memory’ or guidance cues are
left at the site to guide the cell plate towards the mother cell
wall with which it fuses. Besides MTs, actin microfilaments
(MFs), another component of the cytoskeleton, are also known
to play an important role in cytokinesis of plant cells. In particular, the actin-depleted zone (ADZ) was identified as the region
where the cortical MFs disappeared from the division site at the
equator of the cell after disappearance of the PPB-like bands of
MFs in Tradescantia stamen hair cells (Cleary et al. 1992,
Cleary 1995) and in Allium root cells (Liu and Palevitz 1992).
Similarly, in tobacco BY-2 cells, we also found that the ADZ
appeared at the same site where the PPB had existed, and that
the division plane became distorted when the MFs were disrupted by an actin polymerization inhibitor (Hoshino et al.
2003). These findings suggest that the ADZ may receive and
retain some ‘memory’ from the PPB, necessary for determining the division site at cytokinesis.
When the PPB matures by prophase, the bipolar mitotic
spindle of the MTs is formed. However, the exact relationship
between the PPB and spindle poles remains unclear. Generally,
the two poles of the spindle are formed so that the spindle axis
becomes perpendicular to the MTs arrangement of the PPB.
Many reports have indicated that the PPB may relate to the
positioning of spindle poles. In most cases, the directions of the
spindle poles do not change during mitosis. However, in some
cases, for example in the guard mother cells and root apex
cells, the directions of the spindles change at prometaphase,
while the spindle poles are formed in the axis perpendicular to
the PPB at prophase (Wick and Duniec 1984, Mineyuki et al.
1988, Cho and Wick 1989, Cleary and Hardham 1989, Wick
1991, Mineyuki 1999, Scheres and Benfey 1999). In other
reports, some MTs, which connect the PPB and the spindle
poles, were observed (Mineyuki et al. 1991, Nogami et al.
1996). Moreover, monopolar or multipolar spindles were
reported in the endosperm cells at first cell division when the
PPB was never formed (Schmit et al. 1983, Smirnoba and
Bajer 1994, Smirnova and Bajer 1998). From these observations, the PPB was thought to mediate the decision of spindle
pole orientation in prophase.
The PPB is usually identified as a single-ringed band surrounding the nucleus. Although, in some situations, doubleringed PPBs have been reported to appear, these double PPBs
The preprophase band (PPB) of microtubules is
thought to be involved in deciding the future division site.
In this study, we investigated the effects of double PPBs on
spindle formation and the directional decision of cytokinesis by using transgenic BY-2 cells expressing green fluorescent protein (GFP)–tubulin. At prophase, most of the cells
with double PPBs formed multipolar spindles, whereas all
cells with single PPBs formed normal bipolar spindles,
clearly implicating the PPB in deciding the spindle poles. At
metaphase, however, both cell types possessed the bipolar
spindles, indicating the existence of correctional mechanism(s) at prometaphase. From prometaphase to metaphase, the spindles in double PPB cells altered their
directions to become oblique to the cell-elongating axis, and
these orientations were maintained in the phragmoplast
and resulted in the oblique division planes. These oblique
cell plates decreased when actin microfilaments were disrupted, and double actin-depleted zones (ADZs) appeared
where the double PPBs had existed. These results suggest
that the information necessary for proper cytokinesis may
be transferred from the PPBs to the ADZs, even in the case
of the double PPBs.
Keywords: Actin-depleted zone (ADZ) — BY-GT16 cells —
Cytokinesis — Double preprophase bands; Microtubule —
Multipolar spindle.
Abbreviations: ADZ, actin-depleted zone; BA, bistheonellide A;
BY-GT16, BY-2 cell line expressing GFP–tubulin; CB, cytochalasin
B; CD, cytochalasin D; CLSM, confocal laser scanning microscopy;
DAPI, 4′,6-diamidino-2-phenylindole; GFP, green fluorescent protein;
MF, actin microfilament; MT, microtubule; PBS, phosphate-buffered
saline; PPB, preprophase band.
Introduction
At the onset of cell division in higher plants, the phragmosome, a disk-like structure of the cytoplasm, and the pre
prophase band (PPB), a ring of cortical microtubules
(MTs), begin to form and to surround the nucleus (Wick 1991,
Mineyuki 1999, Hoshino et al. 2003). The determination of the
site of cell division is thought to be achieved by the PPB. How1
2
These authors contributed equally to this work.
Corresponding author: E-mail, [email protected]; Fax, +81-4-7136-3706.
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Spindle poles and division plane in double PPBs
Fig. 1 Appearance of multipolar spindles in BY-GT16 and BY-2 double PPB cells. Each focal plane was obtained by CLSM every 1 µm, and
3D images were reconstructed digitally from these optical focal planes (A and B). The single PPB (A) and the double PPBs (B) were observed in
BY-GT16 cells at prophase. BY-GT16 cells were time-sequentially observed from the late G2 phase to metaphase by CLSM (C and D). The cell
with a single PPB (C, 0 min, arrows) developed a (normal) bipolar spindle (C, 8–12 min, spindle poles are indicated by asterisks). In contrast, the
cell with double PPBs (D, 0 min, arrows) first possessed concentrated cores of fluorescence (D, 0 min, arrowheads), then developed a multipolar
spindle at prometaphase (D, 4 min, extra spindle poles are indicated by arrowheads), and finally its spindle altered its form to a bipolar form at
metaphase (D, 35 min). The equatorial plane of the metaphase spindle is indicated by lines (C, 12 min, D, 35 min). The 8-day-old BY-2 cells were
synchronized and then stained with anti-β-tubulin antibody (E-1) and DAPI (E-2). As shown here, the multipolar spindles were also observed in
BY-2 cells. Scale bars indicate 10 µm.
were only transient, being observed for only a brief time from
late G2 phase at prophase, and converged into the single PPB
(Wick and Duniec 1983, Utrilla et al. 1993). Double PPBs
could, however, frequently be induced by synchronization of
elongated BY-2 cells, resulting in oblique phragmoplasts and
cell plates (Hasezawa et al. 1994). Such PPBs remained in their
double form until their disappearance. The question thus arises
that if the PPB determines the formation of spindle poles, then
how are the spindle poles formed in BY-2 cells with double
PPBs? If monopolar or multipolar spindles were formed, then
segregation of the chromosomes would not occur normally.
Furthermore, if the PPBs leave some form of ‘memory’ at the
site to guide the cell plate, then formation of the cell plate
would also not occur normally. In this study, the formation of
the spindle and cell plate was followed in BY-2 cells and BYGT16 cells [a transgenic BY-2 cell line stably expressing a
green fluorescent protein (GFP)–tubulin fusion protein clone
16; Kumagai et al. 2001] that possess double PPBs. As in the
original BY-2 cells, these BY-GT16 cells could be synchronized by drug treatment and could be induced to form double
PPBs. We could then follow the fate of the cells harboring
these double PPBs. The significance of the involvement of
PPBs in the formation of the spindle and division plane, especially in the decision of spindle poles and cell plate, is discussed.
Results
Double PPBs could be induced when 8-day-old elongated
BY-2 cells were synchronized by aphidicolin treatment. In this
experimetal system, about 30% of synchronized 8-day-old BY2 cells could possess the double PPBs in prophase. In our previous report, the formation of oblique cell plates was observed
to bridge two phragmosomes where the double PPBs had
existed (Hasezawa et al. 1994). In this study, we investigated
how the double PPBs give rise to the oblique cell plate. BYGT16 cells were observed by confocal laser scanning microscopy (CLSM) in order to examine spindle formation at
prophase and prometaphase. Even in BY-GT16 cells that possessed double PPBs (Fig. 1B), as with cells that possessed single PPBs (Fig. 1A), the early prophase spindles showed normal
directions that were almost perpendicular to the PPBs. Interestingly, however, it was found that spindles with three or four
poles (designated as multipolar spindles) were formed and preserved from late prophase to prometaphase in most of the cells
that possessed double PPBs (Fig. 1D), whereas normal bipolar
spindles were observed in cells that had possessed single PPBs
(Fig. 1C). In this context, it should be noted that it took 30–
40 min to progress from nuclear envelope breakdown to metaphase in cells with double PPBs (Fig. 1D), which was considerably longer than the 10–20 min in cells with (normal) single
PPBs (Fig. 1C). The origin of the extra poles could already be
identified at prophase to prometaphase as protuberant or concentrated cores of fluorescence (Fig 1D, 0 min, arrowheads).
Therefore, this multipolarity was induced during prophase,
when the double PPBs still existed. Such multipolarity of spindles was also observed in the original BY-2 cells when 8-dayold cells were synchronized as described above and their MTs
were immunostained at prophase or prometaphase with antitubulin antibody (Fig. 1E). Thus, the multipolarity was not
caused by the effects of transformation, and this phenomenon
was not an artifact.
Furthermore, many BY-GT16 cells, harboring double
PPBs, were followed from prophase to prometaphase in order
to examine the relationship between the double PPBs and the
multipolar spindles. In this case, >50 cells in prometaphase
Spindle poles and division plane in double PPBs
Fig. 2 Features of the extra poles in double PPB cells. The BY-GT16
cells were time-sequentially observed at 2 min intervals, and the
number of spindle poles was examined (A). Eighty cells that had a single PPB and 50 cells that possessed double PPBs were examined.
Although the cells with single PPBs never developed any multipolar
spindles (A, single PPB), most of the cells with double PPBs developed multipolar spindles (A, double PPBs). Subsequently, the areas in
which the extra poles first appeared were estimated (B). The region
between the centers of two PPBs was divided into six areas, and these
were designated as the outer, middle and inner areas with respect to their
distance from the PPBs (B-1). Most of the extra poles appeared in the
inner area, which was the central region between the two PPBs (B-2).
from >10 independent synchronization examinations were followed for each cell type; cells that had possessed double PPBs
and those that had possessed the (normal) single PPBs. The
results indicate that multipolarity was usually observed in the
former cell type but scarcely in the latter cell type (Fig. 2A),
suggestive of a clear relationship between double PPBs and
multipolar spindles. In other words, the double PPBs were
most certainly the cause of the extra poles. We subsequently
examined the relationship between the positions of the PPBs
and the extra poles. The nuclear surface was appropriately
divided into six parts between the centers of the two PPBs, and
categorized into three regions; outer, middle and inner, as
shown in Fig. 2B-1. The extra poles, as determined by an
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examination of >40 cells in prometaphase, were localized
mainly in the inner region (Fig. 2B-2). On the other hand, two
original poles were observed necessarily outside of the double
PPBs, and the extra poles were never observed outside of the
double PPBs. This finding suggested that the positions of the
extra poles may not have formed randomly, but were decided
by the PPBs.
Although spindles during prometaphase appeared as
multipolar, the metaphase spindles necessarily showed the
bipolar form (Fig. 1D, 35 min). By closely following the fate of
the extra poles from prophase to metaphase, it became clear
that the extra poles dynamically migrated to their orientations,
and that the multi- (tri-) polar spindles generally changed their
form into the (normal) bipolar spindles during prometaphase
(Fig. 3). Similar results were also observed for the multi- (tetra-)
polar spindles (data not shown). These findings indicated the
existence of some mechanism that could correct the multipolar
spindles into the bipolar spindle forms during prometaphase.
We subsequently observed the BY-GT16 cells in order to
examine the behavior of their mitotic apparatus in detail.
Sequential observations throughout mitosis revealed that the
axis of the spindle changed direction from perpendicular to the
final division plane by metaphase (Fig. 4, 50 min, lines), and
that this changed direction was also maintained in the phragmoplast (Fig. 4, 80 min, lines) and cell plate (Fig. 4, 110 min,
lines). The final division plane was seen to bridge the two
PPBs (Fig. 4, 0 min, 100 min, arrows). In all the cases that we
observed in >80 cells from prophase to early G1 phase, irrespective of the cell plate angles, the altered directions of the
equatorial planes in the metaphase spindles showed agreement
with those of the cell plates. In control cells, the axis of the
spindle, perpendicular to the cell longitudinal axis, did not
change from prophase to metaphase, and was maintained in the
phragmoplast, resulting in the perpendicular cell division plane
(data not shown).
To confirm that cells harboring double PPBs formed
oblique cell plates, we stained the cell plates with aniline blue
and compared their angles in synchronized 8-day-old cell culture, 13 h after the release from aphidicolin, and non-treated 2day-old cell cultures (Fig. 5). We defined the angle of the division plane as that between the cell plate and the cell longitudinal axis, and defined the cell longitudinal axis as the tangent
line of the cell at the site where the cell plate was connected to
the mother cell wall (Fig. 5A, a). In 2-day-old BY-GT16 cells,
almost all the cell plates were transversely orientated to the cell
longitudinal axis (Fig. 5A, a, and B, a), whereas the cell plates
sometimes showed oblique (Fig. 5A, b-1, arrow) or considerably distorted (Fig. 5A, b-2, arrow) orientations in synchronized
8-day-old BY-GT16 cell cultures (Fig. 5B, b). The ‘oblique’
cell plates which were not perpendicular to the focal plane, or
the highly distorted cell plates such as Fig. 5A, b-2, were
counted as ‘unmeasurable’ (UN; Fig. 5B) Approximately 34%
of the cell plates were oblique (or unmeasurable), with angles
<70° to the cell longitudinal axis, in the latter cell lines,
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Spindle poles and division plane in double PPBs
Fig. 3 Time-sequential observations of spindle formation in double PPB cells. A BY-GT16 cell, which possessed double PPBs, was time-sequentially observed by
CLSM from prophase to metaphase. Open circles indicate the PPBs. The nuclear envelope breakdown (NEBD)
occurred at 2 min. An extra spindle pole (arrowhead) was
formed between the two PPBs, then became gradually
translocated, and finally was united into one of the
bipoles (asterisks). As a result, the bipolar spindle was
formed at metaphase. Scale bar indicates 20 µm.
Fig. 4 Time-sequential observations of cell plate formation in double PPB cells. A BY-GT16 cell with double PPBs (0 min, arrows) was followed by CLSM from the late G2 phase to early G1 phase. The direction of the equatorial plane (50 min, lines) at the metaphase spindle was maintained in phragmoplast expansion (80 min, lines) and the division plane (110 min, lines). The two poles maturated at metaphase spindle are
indicated by asterisks. The oblique division plane was formed by connecting the two sites where the PPBs had existed (0 min, 100 min, arrows).
Scale bar indicates 10 µm.
whereas only 3% of the cell plates were oblique in the former
cell cultures.
What were the changes in direction of the mitotic apparatus and their maintenance caused by? When the MFs were
destroyed by bistheonellide A (BA), a dimeric macrolide that
recently was reported to inhibit polymerization of G-actin
(Saito et al. 1998), the typical ‘oblique’ cell plates could not be
induced in cells that had possessed the double PPBs (Fig. 5B,
Spindle poles and division plane in double PPBs
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Fig. 6 Existence of the double ADZs in the double PPB cells. BYGT16 cells were observed by CLSM. While control cell had a single
PPB (A), synchronized 8-day-old cells had double PPBs (B). In this
context, a single ADZ in control cells (C) and double ADZs in synchronized 8-day-old cells (D) were observed, when MFs were stained
with rhodamine–phalloidin. Scale bars indicate 10 µm.
Fig. 5 Angles of the division plane against the cell longitudinal axis.
BY-GT16 cells were stained with aniline blue and were photographed
by fluorescence microscopy (A). The cell longitudinal axis was
defined as the tangent line at the site where the cell plate connected
with the mother cell wall. The angles of the division plane against this
cell longitudinal axis were then measured (A, a, arcuate line) in nontreated 2-day-old (B, a) and in synchronized 8-day-old BY-GT16 cells
(B, b). In the synchronized 8-day-old BY-GT16 cells, the inclined division planes (A, b-1, arrow) and the cells that had non-vertical cell
plates against the field plane (A, b-2, arrow) were increased. The
angles of the latter cell plates were considered as ‘unmeasurable’ (UN)
(B). When MFs were disrupted by BA treatment, the typical ‘oblique’
cell plates could not be induced in cells that had possessed the double
PPBs (B, c), but rather resulted in the appearance of distorted cell
plates (A, c, arrows). Scale bars indicate 20 µm.
c), but rather resulted in the appearance of distorted cell plates
(Fig. 5A, c, arrows). These distorted cell plates were similar to
those of BA-treated cells that had possessed the single PPBs in
our previous report (Hoshino et al. 2003). These data suggested that the decision of cell division planes is controlled by
MFs, even in the case of the double PPBs. It is thought that the
ADZ, which was identified as the region where the cortical
MFs disappeared from the division site at the equator of the
cell, may receive and retain some ‘memory’ from the PPB,
necessary for determining the division site at cytokinesis
(Cleary et al. 1992, Liu and Palevitz 1992, Cleary 1995,
Hoshino et al. 2003). To investigate the structures of MFs in
the cells that had possessed the double PPBs, synchronized 8day-old BY-GT16 cells were stained with rhodamine–phalloidin. While the control cell culture showed a single PPB (Fig.
6A) and single ADZ (Fig. 6C), the double PPBs (Fig. 6B) and
the double ADZs (Fig. 6D) were found in the synchronized 8day-old BY-GT16 cells. These data suggested that in cells with
double PPBs, the existence of the ADZ was also important for
the formation of oblique cell plates as bridges between the two
PPB sites.
Discussion
We previously demonstrated that double PPBs could be
easily induced by synchronizing elongated 8-day-old BY-2
cells, and that cells with the double PPBs showed oblique phragmoplasts and cell plates (Hasezawa et al. 1994). Although, in
some situations, double-ringed PPBs were also reported, they
could only be transiently observed in late G2 phase or prophase,
after which they then converged to single PPBs (Wick and
Duniec 1983, Utrilla et al. 1993). In contrast, the double PPBs
in our current study remained in their double form until their
disappearance. Our investigations into the process of oblique
division plane decision in cells harboring the double PPBs indicated aberrations in the spindle poles and directions.
Multipolar spindle formation in the cells with the double PPBs
The results of our current study suggest that there may be
two stages in the regulation of bipolar spindle formation. The
first stage is that of pole decision by the PPB at prophase, and
the second is that of a correctional mechanism at prometaphase. Concerning the former, it was shown that most cells
harboring double PPBs formed multipolar spindles from
prophase to prometaphase, whereas cells harboring (normal)
single PPBs never formed such multipolar spindles. Thus, the
formation of multipolar spindles is most certainly associated
with the double PPBs, implying that the PPBs at prophase regulate the number of spindle poles. Such multipolar spindles
were also reported in the endosperm cells of higher plants har-
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Spindle poles and division plane in double PPBs
boring no PPBs (Schmit et al. 1983, Smirnova and Bajer 1998),
possibly because the number of spindle poles could not be
decided in the absence of PPBs in them.
The PPB also appears to function in determining the position of the spindle poles. The two poles might be formed at a
certain distance from the PPB. Indeed, most of the extra spindle poles observed in this study appeared at the central area of
the double PPBs. In addition, it was reported that the premitotic nucleus, whose equatorial plane appeared offset from
the (single) PPB, moved to become centered within the PPB
(Mineyuki and Palevitz 1990, Granger and Cyr 2001). These
results may support the idea that the PPBs decide the positions
of the spindle poles at prophase (Wick and Duniec 1984,
Mineyuki et al. 1988, Cleary and Hardham 1989, Cho and
Wick 1989, Wick 1991, Mineyuki 1999). Observations of MTs
that connect the PPB and spindle poles (Mineyuki et al. 1991,
Nogami et al. 1996, Mineyuki 1999) also support the idea that
the PPB decides the pole position, with the MTs functioning
directly to position the spindle poles. Such MTs were also
observed in some of the BY-GT16 cells used in this study.
Moreover, in cells with double PPBs, the MTs were sometimes
observed to connect each PPB to the middle part of the nuclear
surface (data not shown). If such MTs do play a role in deciding the spindle poles, the MTs could account for the appearance
of extra spindle poles between the two PPBs.
Correctional mechanisms of spindles to bipolar forms
If cells progress to metaphase with multipolar spindles,
then chromosomes may not be expected to segregate normally.
In fact, however, prophase cells with extra poles appeared to
form metaphase cells with the normal bipolar spindles, and to
perform normal chromosomal segregation, suggesting that
some correctional mechanism may have functioned at prometaphase. In addition, the time from nuclear envelope breakdown
to metaphase spindle formation was longer in the cells that had
formed the double PPBs than that in the cells with single PPBs.
In the first division of endosperm cells that did not contain a
PPB, some multipolar spindles were identified from prophase
to prometaphase, and only bipolar spindles were observed at
metaphase (Smirnova and Bajer 1998). While the correctional
mechanism may also explain these observations, the actual
processes involved in the mechanism remain unknown.
In most animal cells, the centrosomes become the spindle
poles and form the bipolar spindles. However, even in animal
cells, formation of the bipolar spindles independent of the centrosomes has been reported (Heald et al. 1996, Khodjakov et al.
2000, Wittman et al. 2001). For example, during meiosis of
Xenopus and Drosophila, the bipolar spindles appear without
centrosomes (Gard 1992, Theurkauf and Hawley 1992). Moreover, in an in vitro experimental system using extracts of Xenopus oocytes without centrosomes, formation of the bipolar
spindles around the chromatin-coated beads was reported
(Heald et al. 1996). In these cases, the MT-dependent motor
proteins, kinesin or dynein, appeared to function in formation
of the bipolar spindles (Walczak et al. 1998, Wittman et al.
2001). It was also reported that kif2a, kinesin-related protein
was essential for bipolar spindle assembly during mitosis in
cultured mammalian cells (Ganem and Compton 2003). It may
therefore be possible that the prometaphase correctional mechanism for multipolar spindles of higher plants also involves
these MT-dependent motor proteins. Furthermore, in cells with
double PPBs and multipolar spindles, a greater length of time
was required to form the (normal) metaphase spindle: possibly
for spindle pole correction. These observations suggest that, in
the formation of the bipolar spindle, the formation of a single
PPB is normally the first stage of the regulatory mechanism.
Moreover, even if the bipolar spindle failed to form at this
stage, correction by extra pole movement could be performed
at prometaphase as the second stage of the regulatory mechanism. The mechanism by which the PPB decides the formation
of two poles, and the processes involved in prometaphase correction are issues to be addressed in the future.
Decision of cell division plane by metaphase in the cells with
double PPBs
Although it was reported previously that cells with double PPBs developed oblique phragmoplasts and division planes
(Hasezawa et al. 1994), it is only in this current study that the
directional aberrations were found to have already appeared by
metaphase, and that the equatorial plane of the metaphase spindle was maintained in subsequent phragmoplasts and cell
plates. In the division of Allium guard mother cells, although
the spindle showed an oblique orientation compared with the
PPB at metaphase, the subsequent cell plate was in agreement
with the position where the PPB had existed (Palevitz and Hepler 1974a, Palevitz and Hepler 1974b, Mineyuki et al. 1988).
This suggests that the PPB predicts not the direction of the
metaphase spindle but the position of the cell plate at the telophase phragmoplast. In contrast, all cases we observed in BYGT16 cells (>80 cells) showed agreement with the equatorial
planes of metaphase spindles and cell plates of telophase. However, the prophase spindles of both cell types were perpendicular to the PPBs. This suggests that cells might try to orientate
the spindle perpendicular to the PPB at prophase, but the
appearance of the extra spindle pole and the correction to the
bipolar spindles at prometaphase cause the alteration of the
spindle axis. Mineyuki et al. (1988) reported that early
prophase spindles were oriented perpendicular to the PPB but
gradually became more oblique in prometaphase to metaphase,
and discussed that spindles might change direction because of
space constraints in the cell. According to this hypothesis, it
can be thought that the PPB predicts the position of the equatorial plane of the spindle also in onion guard mother cells, but
space limitation leads to the oblique methaphase spindle in
order to arrange the chromosomes in the equatorial plane. BYGT16 cells seems large enough to have space for the chromosomes to be both perpendicular and oblique to the PPB; this
double PPB-inducing system in BY-GT16 cells may be useful
Spindle poles and division plane in double PPBs
for further investigation how the PPBs determine the direction
of the spindles.
The double ADZ formation in the cells that had double PPBs
In both cases, as the PPBs had already disappeared by
prometaphase, it seems unlikely that the direction would be
directly decided by PPBs or PPB-mediated MTs. In our previous report (Hoshino et al. 2003), we found that the ADZ was
indispensable for organization of the smooth edges of the division plane, possibly by taking over information about the
demarcation of the division site from the PPB and guiding the
edge of the cell plate towards the pre-defined position. The
edges of the cell plate may therefore fuse with the parental
plasma membrane at the correct position at the final stage of
cytokinesis, and this may correspond to stage four of cell plate
formation as described by Samuels et al. (1995). Thus, the
ADZ appears to play an important role in the cytokinesis of
plant cells by preserving the memory of the division site: a
mechanism that may be common in higher plants. In this context, the double PPBs were found to result in double ADZs.
When the double ADZs appeared between prometaphase and
metaphase, after the disappearance of the double PPBs, the
spindles may have been affected by the positional information
retained by the double ADZs, and resulted in the alteration of
their direction to the oblique direction. The phragmoplasts may
also have been guided by this information, and thus formed the
oblique cell plates.
The guidance information on each ADZ appeared to function competitively, since the ‘oblique’ cell plates enlarged
mainly as bridging the sites where two PPBs existed. When the
‘oblique’ cell plates were not perpendicular to the focal plane,
or the planes of the cell plates were highly distorted, these were
counted as ‘unmeasurable’ in the cells with the double PPBs.
Because the ’unmeasurable’ cell plates seldom appeared in
control cells but increased in the synchronized 8-day-old cells,
these ‘unmeasurable’ cell plates could be thought also to be
induced by the double PPBs. The numbers of ‘oblique’ or
‘unmeasurable’ cell plates were increased in the cells with the
double PPBs, and decreased when MFs were disrupted even in
the cells with the double PPBs, indicating that the directional
changes of the division planes were indeed caused by MFs
(double ADZs). The ‘unmeasurable’ cell plates which
remained after actin depolymerizing treatment were similar to
the cell plates in normal single PPB cells whose ADZ was
destroyed by BA (Hoshino et al. 2003), suggesting that the
destruction of the ADZ caused these ‘unmeasurable’ plates in
BA-treated cells.
In conclusion, we investigated the effects of double PPBs
on spindle formation and on determining the orientation of
cytokinesis. First, cells with double PPBs formed multipolar
spindles at prophase. Subsequently, the multipolar spindles
changed into normal bipolar spindles by some correctional
mechanisms at prometaphase. Later, the spindles altered their
directions during the correctional process, and these directions
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at metaphase were maintained in the phragmoplasts and cell
plates, probably because some guidance information was transferred from the double PPBs to the double ADZs.
Materials and Methods
Cell culture and synchronization of tobacco BY-2 cells and BY-GT16
cells
Cell culture and synchronization of tobacco BY-2 and BY-GT16
cell suspensions were performed as described by Nagata et al. (1992).
Briefly, at weekly intervals, suspension cultures of a tobacco BY-2 cell
line, derived from seedlings of Nicotiana tabacum L. cv. Bright Yellow 2, were diluted 95-fold with a modified Linsmaier and Skoog
medium. The 8-day-old cell suspensions were agitated on a rotary
shaker at 130 rpm at 27°C in the dark, and synchronized cells were
obtained. After a 24 h incubation with 5 mg liter–1 aphidicolin, the
cells were thoroughly washed with fresh medium. The cells were then
harvested hourly from 0 to 13 h after the release from aphidicolin to
allow examination of each stage between the S and G1 phase.
A transgenic BY-2 cell line, stably expressing a GFP–tubulin
fusion protein, was previously established and designated as BY-GT16
(Kumagai et al. 2001). This cell line could be maintained and synchronized by almost the same procedures used for the original BY-2 cell
line. In living BY-GT16 cells, the GFP fluorescence, which was localized to MTs, was clearly observable.
Cell staining and observations
For the time sequence observations, 2-day-old or synchronized 8day-old BY-GT16 cells were transferred into 35 mm Petri dishes with
poly-L-lysine-coated, 14 mm coverslip windows on the bottom (Matsunami Glass Ind., Ltd, Osaka, Japan). The dishes were placed onto
the inverted platform of a fluorescence microscope (IX, Olympus Co.
Ltd, Tokyo, Japan) equipped with a confocal laser scanning head and
control systems (GB-200, Olympus).
To stain MTs or MFs, the cells were treated with 250 µM mmaleimidobenzoyl-N-hydroxysuccinimide ester (MBS; Pierce Chemical Co., Rockford, IL, U.S.A.) dissolved in 50 mM PIPES buffer (pH
6.8) containing 1 mM MgSO4, 5 mM EGTA and 1% glycerol (PMEG)
for 30 min prior to fixation (Hasezawa and Nagata 1991). The cells
were fixed with 3.7% (w/v) formaldehyde dissolved in PMEG buffer
for 1 h and were placed onto coverslips coated with poly-L-lysine. The
cells were treated with an enzyme solution containing cellulase Y-C
and pectolyase Y-23 for 5 min, followed by washing with PMEG
buffer. Subsequently, treatment with a detergent solution containing
1% IGEPAL CA-630 was performed for 15 min, followed by washing
with phosphate-buffered saline (PBS; 20 mM Na-phosphate and
150 mM NaCl, pH 7.0). The cells were then treated with a glycine
solution (0.1 M glycine, 1% bovine serum albumin and 0.05% Triton
X-100 in PBS) for 10 min, followed by washing with PBS. The cells
were then stained with a solution of 20 mg litter–1 4′,6-diamidino-2phenylindole (DAPI) for 5 min. To stain MTs, the cells were treated
with mouse anti-β-tubulin antibody (Oncogene Research Products,
Boston, MA, U.S.A.), and then with fluorescein isothiocyanate
(FITC)-conjugated goat anti-mouse IgG secondary antibody (Oncogene) both for 30 min. To stain MFs, cells were incubated with rhodamine–phalloidin (Molecular Probes, Eugene, OR, U.S.A.) for 30 min.
Then, the cells were washed five times with PBS and embedded in a
glycerol solution containing an anti-fading reagent (SlowFade Light,
Molecular Probes). Specimens were examined by CLSM as described
above.
To observe the sites of division where cell plates joined the
parental cell walls, early G1 phase cells were fixed with 3.7% (w/v)
538
Spindle poles and division plane in double PPBs
formaldehyde dissolved in PMEG buffer for 1 h, and stained with
0.05% aniline blue (Biosupplies Australia, Parkville, Victoria, Australia) in PMEG for 30 min. Subsequently, the cells were observed
under a fluorescence microscope (BX51, Olympus).
The images of the cells were processed digitally using Photoshop software (Adobe System Inc., San Jose, CA, U.S.A.).
All procedures described above were performed at room temperature.
Acknowledgments
This study was supported in part by a Grant-in-Aid for Scientific
Research on Priority Areas (Grant No. 15031209) from the Ministry of
Education, Culture, Sports, Science and Technology, Japan, to S.H.
References
Cho, S.O. and Wick, S.M. (1989) Microtubule orientation during stomatal differentiation in grasses. J. Cell Sci. 92: 581–594.
Cleary, A.L. (1995) F-actin redistributions at the division site in living Tradescantia stomatal complexes as revealed by microinjection of rhodamine–phalloidin. Protoplasma 185: 152–165.
Cleary, A.L., Gunning, B.E.S., Wasteneys, G.O. and Hepler, P.K. (1992) Microtubule and F-action dynamics at the division site in living Tradescantia stamen hair cells. J. Cell Sci. 103: 977–988.
Cleary, A.L. and Hardham, A.R. (1989) Microtubule organization during development of stomatal complexes in Lilium rigidum. Protoplasma 149: 67–81.
Ganem, N.J. and Compton, D.A. (2003) The KinI kinesin Kif2a is required for
bipolar spindle assembly through a functional relationship with MCAK. J.
Cell Biol. 166: 473–478.
Gard, D.L. (1992) Microtubule organization during maturation of Xenopus
oocytes: assembly and rotation of the meiotic spindles. Dev. Biol. 151: 516–
530.
Granger, C. and Cyr, R. (2001) Use of abnormal preprophase bands to decipher
division plane determination. J. Cell Sci. 114: 599–607.
Hasezawa, S. and Nagata, T. (1991) Dynamic organization of plant microtubules
at the three distinct transition points during the cell cycle progression of synchronized tobacco BY-2 cells. Bot. Acta 104: 206–211.
Hasezawa, S., Sano, T. and Nagata, T. (1994) Oblique cell plate formation in
tobacco BY-2 cells originates in double preprophase bands. J. Plant Res. 107:
355–359.
Heald, R., Tournebize, R., Blank, T., Sandaltzopoulos, R., Becker, P., Hyman, A.
and Karsenti, E. (1996) Self-organization of microtubules into bipolar spindles around artificial chromosomes in Xenopus egg extracts. Nature 382:
420–425.
Hoshino, H., Yoneda, A., Kumagai, F. and Hasezawa, S. (2003) Roles of actindepleted zone and preprophase band in determining the division site of
higher-plant cells, a tobacco BY-2 cell line expressing GFP-tubulin. Protoplasma 222: 157–165.
Khodjakov, A., Cole, R.W., Oakley, B.R. and Rieder, C.L. (2000) Centrosomeindependent mitotic spindle formation in vertebrates. Curr. Biol. 10: 59–67.
Kumagai, F., Yoneda, A., Tomida, T., Sano, T., Nagata, T. and Hasezawa, S.
(2001) Fate of nascent microtubules organized at the M/G1 interface, as visualized by synchronized tobacco BY-2 cells stably expressing GFP–tubulin:
time-sequence observations of the reorganization of cortical microtubules in
living plant cells. Plant Cell Physiol. 42: 723–732.
Liu, B. and Palevitz, B.A. (1992) Organization of cortical microfilaments in
dividing root cells. Cell Motil. Cytoskel. 23: 252–264.
Mineyuki, Y. (1999) The preprophase band of microtubules: its function as a
cytokinetic apparatus in higher plants. Int. Rev. Cytol. 187: 1–49.
Mineyuki, Y., Marc, J. and Palevitz, B.A. (1988) Formation of the oblique spindle in dividing guard mother cells of Allium. Protoplasama 147: 200–203.
Mineyuki, Y., Marc, J. and Palebitz, B.A. (1991) Relationship between the preprophase band, nucleus and spindle in dividing Allium cotyledon cells. J.
Plant Physiol. 138: 640–649.
Mineyuki, Y. and Palevitz, B.A. (1990) Relationship between preprophase band
organization, F-actin and the division site in Allium. Fluorescence and morphometric studies on cytochalashin-treated cells. J. Cell Sci. 97: 283–295.
Nagata, T., Nemoto, Y. and Hasezawa, S. (1992) Tobacco BY-2 cell line as the
‘Hela’ cell in the cell biology of higher plants. Int. Rev. Cytol. 132: 1–30.
Nogami, A., Suzaki, T., Nagahama, Y. and Mineyuki, Y. (1996) Effects of
cycloheximide on preprophase bands and prophase spindles in onion (Allium
cepa L.) root tip cells. Protoplasma 192: 109–121.
Palevitz, B.A. and Hepler, P.K. (1974a) The control of the plane of division during stomatal differentiation in Allium. I. Spindle reorientation. Chromosoma
46: 297–326.
Palevitz, B.A. and Hepler, P.K. (1974b) Control of plane of division during stomatal differentiation in Allium. II. Spindle reorientation. Chromosoma 46:
327–341.
Saito, S., Watabe, S., Ozaki, H., Kobayashi, M., Suzuki, T., Kobayashi, H.,
Fusetani, N. and and Karaki, H. (1998) Actin-depolymerizing effect of
dimeric macrolides, bistheonellide A and swinholide A. J. Biochem. 123:
571–578.
Samuels, A.L., Giddings, T.H. and Staehelin, L.A. (1995) Cytokinesis in
tobacco BY-2 and root tip cells: a new model of cell plate formation in higher
plants. J. Cell Biol. 130: 1345–1357.
Scheres, B. and Benfey, P.N. (1999) Asymmetric cell division in plants. Annu.
Rev. Plant Physiol. Plant Mol. Biol. 50: 505–537.
Schmit, A.C., Vantard, M., Mey, J. and Lambert, A.M. (1983) Aster-like microtubule centers establish spindle polarity during interphase–mitosis transition
in higher plant cells. Plant Cell Rep. 2: 285–288.
Smirnoba, E.A. and Bajer, A.S. (1994) Microtubule converging centers and
reorganization of the interphase cytoskeleton and the mitotic spindle in higher
plant Haemanthus. Cell Motil. Cytoskel. 27: 219–233.
Smirnova, E.A. and Bajer, A.S. (1998) Early stages of spindle formation and
independence of chromosome and microtubule cycles in Haemanthus
endosperm. Cell Motil. Cytoskel. 40: 22–37.
Theurkauf, W.E. and Hawley, R.S. (1992) Meiotic spindle assembly in Drosophila females: behavior of nonexchange chromosomes and the effects of
mutations in the nod kinesin-like protein. J. Cell Biol. 116: 1167–1180.
Utrilla, L., Giménez-Abián, M.I. and Torre, C.D. (1993) Timing the phases of
the microtubule cycles involved in cytoplasmic and nuclear divisions in cells
of undisturbed onion root meristems. Biol. Cell 78: 235–241.
Walczak, C.E., Vernos, I., Mitchison, T.J., Karsenti, E. and Heald, R. (1998) A
model for the proposed roles of different microtubule-based motor proteins in
establishing spindle bipolality. Curr. Biol. 8: 903–913.
Wick, S.M. (1991) The preprophase band. In The Cytoskeletal Basis of Plant
Growth and Form. Edited by Lloyd, C.W. Academic Press, London, pp. 231–
244.
Wick, S.M. and Duniec, J. (1983) Immunofluorescence microscopy of tubulin
and microtubule arrays in plant cells. I. Preprophase band development and
concomitant appearance of nuclear envelope-associated tubulin. J. Cell Biol.
97: 235–243.
Wick, S.M. and Duniec, J. (1984) Immunofluorescence microscopy of tubulin
and microtubule arrays in plant cells. II. Transition between the pre-prophase
band and the mitotic spindle. Protoplasma 122: 45–55.
Wittman, T., Hyman, A. and Desai, A. (2001) The spindle: a dynamic assembly
of microtubules and motors. Nat. Cell Biol. 3: E28–E34.
(Received June 9, 2004; Accepted January 7, 2005)