Download Time-Sequence Observations of Microtubule Dynamics throughout

Plant CellPhysiol. 41(2): 244-250 (2000)
JSPP © 2000
Rapid Report
Time-Sequence Observations of Microtubule Dynamics throughout Mitosis
in Living Cell Suspensions of Stable Transgenic Arabidopsis—Direct
Evidence for the Origin of Cortical Microtubules at M/Gi Interface—
Seiichiro Hasezawa1, Katsumi Ueda 2 and Fumi Kumagai1
1
2
Department of Integrated Biosciences, Graduate School of Frontier Sciences, The University of Tokyo, Hongo, Bunkyo-ku, Tokyo,
113-0033 Japan
Department of Biological Science, Nara Women's University, Nara, 630-8263 Japan
Transgenic Arabidopsis thaliana, stably expressing a
GFP-TUA6 fusion protein, were subcultured in B5 medium supplemented with 2,4-D and BA. In the cell suspensions, the microtubular changes in the mitotic cells could
be monitored by time-sequence observations using a timelapse system of fluorescence microscopy. We have succeeded in following the microtubule (MT) dynamics in living cells throughout mitosis, from the late G2 phase to early
d phase, and found that, at the M/Gi interface, the cortical MTs were firstly reorganized in the perinuclear regions
and then in the cortex, as we had previously suggested
(Hasezawa and Nagata 1991, Nagata et al. 1994). The significance of this observation on the origin of cortical MTs
is discussed.
Key words: Arabidopsis — GFP-TUA6 fusion protein —
Microtubule — M/Gi interface — Suspension culture —
Transgenic plants.
In plants, microtubules (MTs) play an important role
in cell morphogenesis and show dynamic changes in their
distribution during cell cycle progression. Three types
of MT distribution, specific to plant cells, appear in the
following order; cortical MTs that control cellulosemicrofibril deposition in the cell wall, the preprophase
band (PPB) MTs that mark the future division site, and
phragmoplast MTs that produce the cell plate. Such
changes in the distribution of MTs have been studied by
immuno-cytochemical techniques using anti-tubulin antibodies on fixed plant cells (Lloyd 1987, Staiger and Lloyd
1991, Goddard et al. 1994). However, despite the considerable amount of valuable information about MT dynamics obtained from such studies, some of the transitional processes in MT formation are still unclear and
several controversies, for example regarding the formation
of the PPB or the origin of cortical MTs, still remain.
Abbreviations: BA, 6-benzylaminopurine; GFP, green fluorescent protein; MT, microtubule; PPB, preprophase band.
244
Recent studies by microinjection of tubulins conjugated with fluorescent dyes have revealed the reorientation
of cortical MTs in living cells of Tradiscantia stamen hairs
and the pea epidermis (Zhang et al. 1990, Wasteneys et al.
1993, Yuan et al. 1994, 1995, Wymer et al. 1997). This
technique, however, requires special skills and is rather
limited in the materials that can be used. In the development of a more convenient technique, the green fluorescent
protein (GFP) was recently employed to observe MT reorientation by transient expression of a GFP-MAP4 fusion
protein in Fava Bean cells transformed by particle bombardment (Marc et al. 1998). In our previous study, stable
transformants of Arabidopsis thaliana, which continuously expressed a GFP-TUA6 fusion protein, were established,
and the GFP-fluorescence of MTs could be detected in the
cells of the leaf and hypocotyl (Ueda et al. 1999). In that
study, the figures showing the dynamics of MT reorientation appeared to be intact and reliable, since the GFP
protein associated directly with the tubulin protein. Actually, the fibers visualized by GFP fluorescence corresponded to the MTs stained by the anti-tubulin antibody
(Ueda et al. 1999). Thus, the GFP technique appears to be
useful for following MT dynamics, yet, in the studies described above, MT dynamics during mitosis, at which time
the most dramatic changes in MTs occur, have not yet been
observed. This may be due to the low frequency of mitotic
cells that occur in the total cell populations. Therefore, in
this study, we attempted to establish a cell line of stable
Arabidopsis transformant, expressing a GFP-TUA6 protein, in which MT dynamics during mitosis could be observed. For our purpose, a fine cell culture, that could grow
vigorously and which showed bright GFP-fluorescence of
MTs, was required. Such a cell line was established as follows.
Establishment and observations of an Arabidopsis cell
suspension expressing the GFP-tubulin fusion protein—
Seedlings of transformants (Ueda et al. 1999), harvested 10
d after the germination of sterilized seeds, were cut and
placed onto Murashige and Skoog (1962) agar medium
supplemented with 1 mg liter"1 2,4-D and 0.1 mg liter 1
kinetin. After a month, the calli derived from the seedling
Microtubules at M/Gi interface in living cells
245
Fig. 1 Time-sequence observations of the dynamic changes in the mitotic apparatus of an AGT cell by a time-lapse system throughout
mitosis. The scans were performed at 10-min intervals, for a total of over 2 h. 0-10 min, PPB was developing; 20-30 min, spindle was
changing from prometaphase to anaphase; 40-50 min, phragmoplast was growing; 60-70 min, short MTs were formed in the perinuclear
region; 80-110 min, cortical MTs were gradually organized and the daughter nuclei were separating from each other. Bar represents 10/urn.
246
Microtubules at M/Gi interface in living cells
strips were transferred into a liquid B5 medium (Gamborg
et al. 1968) supplemented with 1 mg liter"1 2,4-D and 1 mg
liter"1 BA. The cell suspensions were repeatedly filtrated
through 1-mm pore-sized meshes in order to produce fine
cell cultures suitable for observing GFP-labeled-MTs of the
cells. After an additional month, the cell line most suitable
for MT observations was selected by monitoring the GFPfluorescent cells by fluorescence microscopy at 488 nm excitation. Finally, a cell line was established and designated
as AGT {Arabidopsis cell suspension expressing GFPTubulin fusion protein). The AGT suspensions were
maintained in the B5 medium described above by transferring 10 ml of culture into 80 ml fresh medium every two
weeks and culturing on a rotary shaker at 130 rpm at 27 °C
under continuous light. In living AGT cells, GFP fluorescence, localized at MTs, was clearly observable. For timesequence observations, AGT cells, three days after transfer, were retransferred into 035 mm Petri dishes with 014
mm coverslip windows at the bottom (Matsunami Glass
Ind., Ltd., Osaka, Japan). The dishes were placed onto the
inverted platform of an 1X70 microscopy system for timelapse analysis with a cooled-CCD camera (MicroMAX/
OL, Olympus Japan Co., Ltd.). Although initially evaluated various systems, such as CLSM, that would allow us
to observe AGT cells with the minimum account of damage, the system used here, as described above, was finally
found to be the most ideal for our purpose.
Dynamics of MT changes from late G2 phase to early
G] phase—Using the GFP technique, we could follow, for
the first time in either higher plant or animal cells, the MT
dynamics of the mitotic apparatus in a living cell from the
late G2 phase to the early Gi phase (Fig. 1). Each scan of an
AGT cell was performed for 15 s at 10-min intervals using
a x40 dry objective (SLCPlanFl, Olympus). The PPB,
spindle and phragmoplast were clearly recognizable, in
order, within the 0-10, 20-30 and 40-50 min periods after
the start of the time-sequence observations, respectively.
Subsequently, the short MTs began to be actively formed
in the perinuclear region, the cortical MTs became reorganized, and thereafter, the daughter nuclei began to separate from each other. In this case, the total observation
time was 2 h, and the period of M phase was estimated at
about 1 h. As this period closely corresponded to that of
control Arabidopsis cells, we concluded that the observed
cell had not been injured by the fluorescence used for the
observations, and that the cell cycle progression had not
been blocked.
Events at the M/Gj interface in AGT cells—From the
above observations, the formation of the short MTs in the
perinuclear region was determined to be most prominent at
the 60-70 min period (Fig. 1). We then attempted to perform more detailed observations in order to clarify the
mechanism by which cortical MTs become reorganized at
the M/Gi interface. For this purpose, we followed the cell
cycle progression of an AGT cell from the anaphase to the
early Gi phase. Each scan of the cell was performed for 0.5
s at 2-min intervals using a x 60 oil objective (PlanApo
oil LSM, Olympus). The phragmoplast was organized
between the rests of the spindles of both poles at late
anaphase (Fig. 2, 0-2 min), continued growth for about
another 15 min, and finally reached the lateral cell wall of
the mother cell (Fig. 2, 2-14 min). Subsequently, the signs
of MTs became recognizable near the nuclear envelopes of
the two daughter nuclei (Fig. 2, 16-22 min), after which
numerous short MTs began to form in the perinuclear
region as the phragmoplast began to degenerate. Thus
short MTs appeared to be formed at the expense of the
phragmoplast. The MT signals were initially observed
mainly in areas between the nuclei and division site (Fig. 2,
24-36 min), and the short MTs then gradually elongated to
the lateral cortex (Fig. 2, 38-48 min). Finally, the MTs became reorganized throughout the cortex, together with the
separation of both nuclei (Fig. 2, 50-58 min).
We also attempted to observe the reorganization of
cortical MTs on the cell cortex. For this purpose, we followed the cell cycle progression of an AGT cell from the
organization of MTs on the perinuclear region at M/Gi
interface. The short MTs from the nuclei (Fig. 3, 3 min)
and the sign of the cortical MTs (Fig. 3, 4 min) were
recognizable in order. Subsequently, the cortical MTs were
gradually reorganized (Fig. 3, 6-14 min).
Significance of the observation system using AGT
cells—In this study we have shown, for the first time, the
dynamics of MTs during the mitotic process, and the reorganization of cortical MTs at the M/Gi interface in living, higher plant cells. These results provide direct evidence
for our hypothesis that the cortical MTs are first formed in
the perinuclear regions, and then become organized in the
cell cortex near the division site (Hasezawa et al. 1991,
1997, Nagata et al. 1994, Kumagai et al. 1995, 1999). Moreover, our current results, taken together with our earlier
observations on fixed tobacco BY-2 cells, argue for a
general mechanism by which cortical MTs are reorganized
in higher plant cells. In addition, the AGT cell suspension
of the stable transformant expressing a GFP-TUA6 fusion
protein, used here, showed considerable mitotic figures
within a few days after the transfer, and could constantly
provide a source of cells for time-sequence observations of
MT dynamics whenever required. In the current observation system, MT changes during the cell cycle could be
monitored by real-time observations. However, this system
could be greatly improved, if synchronization of cell cycle
progression, as in BY-2 cells (Nagata et al. 1992), could be
introduced into the AGT cells.
In summary, this is the first report, for both higher
plant and animal cells, in which the GFP technique was
used to follow, in detail, the MT dynamics from the late
G2 phase to the early Gi phase. The AGT cell suspensions
Microtubules at M/Gi interface in living cells
247
Fig. 2 Time-sequence observations from the anaphase to the early Gi phase. The scans were performed at 2-min intervals. The dynamic changes in the phragmoplast MTs, the short MTs, and the cortical MTs could be observed in detail. Arrow heads show the
division plane. Bar represents lOjum. N: nucleus.
248
Microtubules at M/Gi interface in living cells
Microtubules at M/Gi interface in living cells
249
Fig. 3 Time-sequence observations at M/Gi interface. The scans were performed on the cell cortex. The reorganization of the cortical
MTs could be observed in detail. Arrow (4 min) shows the sign of the cortical MTs. Arrow heads show the division plane. Bar represents
N: nucleus.
Microtubules at M/Gi interface in living cells
250
used here, may well serve as novel and useful tools for
studies into cytoskeletal events during cell cycle progression in higher plant cells.
The authors thank Drs. H. Fukuda and N. Kondo of the
University of Tokyo for providing the MicroMax/OL system, and
for critical reading of the manuscript, respectively. This study was
supported in part by a Grant-in-Aid from the Ministry of Education, Science, Sports and Culture, Japan to S.H.
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(Received December 1, 1999; Accepted December 29, 1999)